Editing system and its components and their use

Abstract

Guide nucleic acid conjugates, systems comprising guide nucleic acid conjugates, and methods for using them are provided. Such guide nucleic acid conjugates and systems are useful for gene editing and epigenetic regulation of gene expression.

Description

[Technical Field]

[0001] Sequence List This application is filed together with an electronic sequence listing. The sequence listing is provided as a file named EDIT0001SEQ.xml, created on September 13, 2024, with a size of 2,646 KB. The electronic information of this sequence listing is incorporated herein by reference in its entirety.

[0002] This embodiment provides a guided nucleic acid binding agent such as a Cas protein, a guide, an editing system including the guided nucleic acid binding agent and the guide, and a method for using them. [Background technology]

[0003] Cas proteins, along with their associated clustered, regularly arranged short palindromic repeat (CRISPR) guided ribonucleic acid (gRNAs), appear to be ubiquitous components of the prokaryotic immune system (approximately 45% of bacteria and 84% of archaea). These CRISPR-gRNA cleavage systems help protect such microorganisms from non-self nucleic acids such as infectious viruses and plasmids. CRISPR-associated (Cas) proteins are highly diverse and contain a wide variety of nucleic acid-binding domains. Cas nucleic acid-binding domains can be utilized for a diverse range of applications, including, but not limited to, gene editing and epigenetic regulation of gene expression. [Overview of the project]

[0004] Certain embodiments provided herein relate to guide nucleic acid binders (Cas proteins, dead Cas proteins, and Cas fusion proteins), guides (single guides and dual guides), and editing systems comprising such guide nucleic acid binders and guides. Certain embodiments relate to methods of using such editing systems.

[0005] In certain embodiments, polypeptides are provided herein. In certain embodiments, polypeptides are guide nucleic acid binders. In certain embodiments, polypeptides provided herein are Cas proteins. In certain such embodiments, Cas proteins are Cas enzymes that can induce cleavage of both strands of a double-stranded DNA target (double-strand breaks). In certain embodiments, Cas proteins are Cas enzymes that can induce cleavage of one strand of a double-stranded DNA target (nicks). In certain embodiments, Cas proteins are dead Cas proteins that do not induce cleavage of double-stranded DNA. In certain embodiments, polypeptides comprise Cas proteins. Certain Cas proteins can be fused with one or more other proteins or protein domains, also known as "heterogeneous domains," to construct Cas fusion proteins.

[0006] In certain embodiments, nucleic acids encoding the polypeptides described above are provided herein. In certain embodiments, such nucleic acids are DNA (e.g., template DNA) or RNA (e.g., mRNA).

[0007] In certain embodiments, guides are provided herein. In certain embodiments, the guide consists of a single oligonucleotide (single guide). In certain embodiments, the guide consists of two oligonucleotides (a first oligonucleotide and a second oligonucleotide) that are partially complementary to each other and allow them to hybridize together (dual guide). The guide includes a target recognition region and a protein recognition region. In the case of a single guide, the target recognition region and the protein recognition region are regions of a single oligonucleotide. In the case of a dual guide, it consists of a first oligonucleotide and a second oligonucleotide, where the first oligonucleotide includes a first portion of the target recognition region and the protein recognition region, and the second oligonucleotide includes a second portion of the protein recognition region. In certain such embodiments, the first oligonucleotide may be called "crRNA" and the second oligonucleotide may be called "tracrRNA". The target recognition region of the guide is a region of an oligonucleotide (either the region of a single guide or the region of crRNA in the case of a dual guide) having a nucleic acid base sequence complementary to an isolength portion of the target sequence of the target nucleic acid. In certain embodiments, the target nucleic acid is target DNA.

[0008] In certain embodiments, the editing system includes a guide nucleic acid binder and a guide. In certain such embodiments, the guide nucleic acid binder is a Cas protein. In certain embodiments, the guide is a single guide. In certain embodiments, the editing system includes a Cas protein and a single guide.

[0009] In certain embodiments, compositions comprising template DNA encoding both a guide nucleic acid binder and a guide are provided herein. In certain embodiments, compositions comprising template DNA encoding both a Cas protein and a guide are provided herein. In certain embodiments, compositions comprising mRNA encoding a guide and a guide nucleic acid binder are provided herein. In certain embodiments, compositions comprising mRNA encoding a guide and a Cas protein are provided herein. In certain embodiments, compositions comprising mRNA encoding a single guide and a guide nucleic acid binder are provided herein. In certain embodiments, compositions comprising mRNA encoding a dual guide and a guide nucleic acid binder are provided herein. In certain embodiments, compositions comprising mRNA encoding a single guide and a Cas protein are provided herein. In certain embodiments, compositions comprising mRNA encoding a dual guide and a Cas protein are provided herein. In such embodiments, the Cas protein is selected from Cas enzymes, dead Cas proteins, and Cas fusion proteins. In certain embodiments, such compositions are administered to a subject to induce gene editing. [Brief explanation of the drawing]

[0010] [Figure 1] This shows a gel demonstrating the cleavage activity of the ION-CAS protein in HEK293T nuclear extract, using a general-purpose target recognition region as a guide. [Figure 2] This gel demonstrates the cleavage activity of the ION-CAS protein in HEK293T nuclear extract, using Cas-specific target recognition regions and Cas-specific protein recognition regions in guide RNA. [Figure 3] The consensus PAM sequences for IONCAS008, IONCAS009, and IONCAS0016 are shown. [Figure 4a] This shows a gel demonstrating the cleavage activity of the ION-CAS protein in HEK293T nuclear extract, using a general-purpose target recognition region as a guide. [Figure 4b] This shows a gel demonstrating the cleavage activity of the ION-CAS protein in HEK293T nuclear extract, using a general-purpose target recognition region as a guide. [Figure 4c] This shows a gel demonstrating the cleavage activity of the ION-CAS protein in HEK293T nuclear extract, using a general-purpose target recognition region as a guide. [Figure 5] This provides a schematic diagram of the Cas editing system. [Figure 6] This provides a schematic diagram for labeling the secondary structure of the SpCas9 guide. [Modes for carrying out the invention]

[0011] It should be understood that both the above summary and the following detailed description are illustrative and descriptive only and do not limit the embodiments as claimed. In this specification, the use of the singular includes the plural unless otherwise explicitly stated. Where used herein, the use of “or” means “and / or” unless otherwise specified. Furthermore, the use of the term “including,” as well as other forms such as “includes” and “included,” is not limiting.

[0012] The section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein. All documents or parts of documents referenced herein, including, but not limited to, patents, patent applications, articles, books, and papers, as well as GenBank and NCBI reference sequencing records, the parts of documents considered herein, and all of them, are expressly incorporated herein by reference.

[0013] Throughout this specification, unless otherwise indicated, the first letter of a peptide sequence is understood to be the first amino acid of the N-terminal peptide, and the last letter of a peptide sequence is understood to be the last amino acid of the C-terminal peptide. Similarly, unless otherwise indicated, the first nucleoside in a nucleotide sequence represents the 5' end of the nucleotide, and the last letter in a nucleotide sequence represents the 3' end.

[0014] Unless otherwise specified, the nomenclature, procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and medicinal chemistry and pharmaceutical chemistry described herein are well known and commonly used in the art. Where permitted, all patents, patent applications, patent application publications, and other publications and data referenced throughout this disclosure are incorporated herein by reference in their entirety.

[0015] Unless otherwise specified, the following terms have the meanings set forth below.

[0016] As used herein, “2'-deoxynucleoside” means a nucleoside containing a 2'-H(H)deoxyfuranosyl sugar moiety. In certain embodiments, the 2'-deoxynucleoside is a 2'-β-D-deoxynucleoside, which contains a 2'-β-D-deoxyribosyl sugar moiety having a β-D ribosyl configuration as found in naturally occurring deoxyribonucleic acid (DNA). In certain embodiments, the 2'-deoxynucleoside may contain a modified nucleic acid base or an RNA nucleic acid base (uracil).

[0017] As used herein, "2'-F" refers to a 2'-F group replacing the 2'-OH group in the furanosyl sugar moiety. "2'-fluoro sugar moiety" or "2'-F sugar moiety" refers to a sugar moiety having a 2'-F group instead of the 2'-OH group in the furanosyl sugar moiety. Unless otherwise indicated, the 2'-F sugar moiety is in a β-D-ribosyl configuration.

[0018] As used herein, "2'-F nucleoside" means a nucleoside containing a 2'-F sugar moiety.

[0019] As used herein, “2'-MOE” means a 2'-OCH2CH2OCH3 group in place of the 2'-OH group in the furanosyl sugar moiety. “2'-MOE sugar moiety” means a sugar moiety having a 2'-OCH2CH2OCH3 group in place of the 2'-OH group in the furanosyl sugar moiety. Unless otherwise indicated, the 2'-MOE sugar moiety is in a β-D-ribosyl configuration. “MOE” means O-methoxyethyl.

[0020] As used herein, "2'-MOE nucleoside" means a nucleoside containing a 2'-MOE sugar moiety.

[0021] As used herein, "2'-NMA" means a 2'-O-CH2-C(=O)-NH-CH3 group instead of the 2'-OH group in the ribosyl sugar moiety. A "2'-NMA sugar moiety" is a sugar moiety having a 2'-O-CH2-C(=O)-NH-CH3 group instead of the 2'-OH group in the ribosyl sugar moiety. Unless otherwise indicated, the 2'-NMA sugar moiety is in a β-D configuration. "NMA" means O-(N-methyl)acetamide.

[0022] As used herein, "2'-NMA nucleoside" means a nucleoside containing a 2'-NMA sugar moiety.

[0023] As used herein, "2'-OMe" means a 2'-OCH3 group instead of a 2'-OH group in the furanosyl sugar moiety. "2'-O-methyl sugar moiety" or "2'-OMe sugar moiety" means a sugar moiety having a 2'-OCH3 group instead of a 2'-OH group in the furanosyl sugar moiety. Unless otherwise indicated, the 2'-OMe sugar moiety is in a β-D-ribosyl configuration.

[0024] As used herein, "2'-OMe nucleoside" means a nucleoside containing a 2'-OMe sugar moiety.

[0025] As used herein, “2'-substituted nucleoside” means a nucleoside containing a 2'-substituted sugar moiety. As used herein, with respect to the sugar moiety, “2'-substituted” means a sugar moiety containing at least one 2'-substituent other than H or OH.

[0026] As used herein, "5-methylcytosine" means cytosine modified with a methyl group attached at the 5-position. 5-methylcytosine is a modified nucleic acid base.

[0027] As used herein, “active site” means the region of a protein to which a substrate molecule binds and which undergoes enzymatic catalysis of a chemical reaction. An active site comprises one or more catalytic residues. As used herein, “catalytic residue” means an amino acid that is involved in the enzymatic catalysis of a chemical reaction at that active site. A catalytic residue may be directly involved in the catalytic mechanism (e.g., as a nucleophile), may exert an effect on another residue or water molecule directly involved in the catalytic mechanism that assists the catalytic action (e.g., by electrostatic or acid-base action), may stabilize a transition state intermediate, and / or may exert an effect on a substrate or cofactor that assists the catalytic action, including steric and electrostatic effects (e.g., by polarizing the bond being cleaved).

[0028] As used herein, “Cas enzyme” means a guide nucleic acid binder capable of cleaving one or both strands of double-stranded DNA. In certain embodiments, the target nucleic acid is double-stranded “target DNA.” The “Cas enzyme” may be an enzyme capable of cleaving both strands of double-stranded DNA, or it may be an enzyme capable of cleaving only one strand of double-stranded DNA (“Cas nickase”) (for example, containing a mutation in one of the catalytic residues but not in both).

[0029] As used herein, “Cas protein” means “Cas enzyme” or “dead Cas protein.”

[0030] As used herein, “dead Cas protein” means a guide nucleic acid binder that cannot cleave either strand of double-stranded DNA.

[0031] As used herein, "cEt" or "restricted ethyl" means a bicyclic sugar moiety, where the first ring of the bicyclic sugar moiety is a ribosyl sugar moiety, and the second ring of the bicyclic sugar is formed via a bridge connecting the 4'-carbon and 2'-carbon, the bridge having the formula 4'-CH(CH3)-O-2', and the bridge is in the S configuration. The cEt bicyclic sugar moiety is in the β-D configuration.

[0032] As used herein, “cleavable portion” means a bond or group of atoms that is cleaved under physiological conditions, for example, within a cell or object.

[0033] As used herein, “complementary” with respect to an oligonucleotide means that at least 70% of the nucleic acid bases of that oligonucleotide and the nucleic acid bases or one or more regions of another nucleic acid can form hydrogen bonds with each other when the nucleic acid base sequences of that oligonucleotide and the other nucleic acid are aligned in opposite directions. “Complementary region” with respect to a region of an oligonucleotide means that at least 70% of the nucleic acid bases of that region and the nucleic acid bases or one or more regions of another nucleic acid can form hydrogen bonds with each other when the nucleic acid base sequences of that oligonucleotide and the other nucleic acid are aligned in opposite directions. Complementary nucleic acid bases mean nucleic acid bases that can form hydrogen bonds with each other. Complementary nucleic acid base pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and / or nucleic acids do not need to have nucleic acid base complementarity at each nucleoside. Rather, some mismatches are acceptable. As used herein, “fully complementary” or “100% complementary” with respect to an oligonucleotide means that the oligonucleotide is complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.

[0034] As used herein, the “complementary strand” of target DNA is a strand of double-stranded DNA that is complementary to the target recognition region of the guide. As used herein, the “non-complementary strand” of target DNA is a strand of double-stranded DNA that is complementary to the “complementary strand.” The PAM sequence is found within the “non-complementary strand.”

[0035] As used herein, “domain” means a subset of amino acids linked together in a polypeptide. A domain may consist of several smaller domains called subdomains; for example, the RuvC domain of Streptococcus pyogenes SpyCas9 consists of three non-contiguous subdomains: RuvC-I, RuvC-II, and RuvC-III.

[0036] As used herein, “double-stranded break,” “double-strand break,” or “DSB” refers to a break in both strands of double-stranded DNA.

[0037] As used herein, “exogenous mRNA” means any mRNA that has been introduced into an organism or cell and has not been synthesized by the recipient organism or cell itself. Exogenous mRNA can be isolated or purified from an organism or cell, transcribed in vitro, or produced by synthetic means. Exogenous mRNA contains a coding region (e.g., an open reading frame (ORF)) that encodes a polypeptide sequence.

[0038] As used herein, “gene editing,” “genome editing,” or “genomic editing” means a process of altering the nucleic acid base sequence of a genome (e.g., insertion, deletion, mutation, or alteration of nucleic acid bases, including epigenetic states (e.g., A or G methylation states)) directly or via innate cellular processes, for example, after the introduction of a double-strand break or nick. In certain embodiments, gene editing is mediated by a complex comprising a guide nucleic acid binder and a guide. In certain embodiments, gene editing is mediated by a complex comprising a Cas protein, a dead Cas protein, or a Cas fusion protein and a guide.

[0039] As used herein, “guide” means either an oligonucleotide (“single guide”) or a complex of two oligonucleotides that are partially hybridized with each other (“dual guide”), in either case including a target recognition region and a protein recognition region. In embodiments where the guide is a single guide, the target recognition region and the protein recognition region are regions of one oligonucleotide constituting the single guide. In embodiments where the guide is a dual guide, the target recognition region is a region of a first oligonucleotide, and the protein recognition region is a portion of a complex containing regions of each of the two oligonucleotides. In certain embodiments, the guide directs the guide nucleic acid binder to a target sequence of target DNA.

[0040] As used herein, “guide nucleic acid conjugate” means a polypeptide comprising (1) a region that interacts with the protein recognition region of the guide, and (2) a region that interacts with the PAM of the target DNA. In certain embodiments, the target recognition region of the guide causes the guide nucleic acid conjugate to specifically interact with the target DNA. The guide nucleic acid conjugate may comprise one or more active or inactive nuclease domains. The guide nucleic acid conjugate may comprise heterologous domains. Guide nucleic acid conjugates include Cas enzymes, dead Cas proteins, and Cas fusion proteins.

[0041] As used herein, “HNH domain” or “HNH” means a cation-dependent endonuclease domain of a protein having an active site containing a histidine (H) catalytic residue. In certain embodiments, the active site of the HNH domain contains histidine (H) and asparagine (N) catalytic residues. In certain embodiments, the HNH domain of a Cas protein cleaves the complementary strand of target DNA. The HNH domain may have one or more substitutions that catalytically inactivate it.

[0042] As used herein, “hybridization” means the annealing of oligonucleotides and / or nucleic acids. While not limited to a specific mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding between complementary nucleic acid bases. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, antisense compounds and nucleic acid targets.

[0043] As used herein, "identity" or "percent identity" with respect to amino acid sequences means the percentage of amino acids that are identical between two amino acid sequences when the amino acid sequences are aligned by the greatest similarity. Percent identity can be determined by programs such as BLASTP (Altschul, et al., J.Mol.Biol., 1990; Altschul, et al., Nucleic Acids Research, 1997) or Clustal Omega (Sievers, et al., Molecular Sys.Biol., 2011; Goujon et al., Nucleic Acids Research, 2010; McWilliam, et al., Nucleic Acids Research 2013).

[0044] As used herein, the term “interacts with” with respect to two macromolecules (e.g., a polypeptide and an oligonucleotide; two polypeptides bound to each other; or a polypeptide and a nucleic acid) means that multiple non-covalent stabilizing interactions are formed between the two macromolecules, including but not limited to bipolar interactions (hydrogen bonding), cation-π interactions or π-π stacking interactions, electrostatic interactions (salt bridges), and hydrophobic interactions.

[0045] As used herein, the term “nucleoside bond” refers to a covalent bond between adjacent nucleosides within an oligonucleotide. As used herein, “modified nucleoside bond” means any nucleoside bond other than a phosphodiester nucleoside bond. As used herein, “mismatched” or “non-complementary” means a nucleic acid base of the first oligonucleotide that is not complementary to the corresponding nucleic acid base of the second oligonucleotide or the target nucleic acid when the first and second oligonucleotides are aligned.

[0046] As used herein, “motif” means a pattern of unmodified and / or modified sugar moieties, nucleic acid bases, and / or nucleoside bonds in an oligonucleotide.

[0047] "Modification guide" refers to a guide containing modified nucleoside bonds, modified sugar moieties, and / or modified nucleic acid bases.

[0048] As used herein, “natural amino acids” means Gly or each of the following L - Represents isomers: Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val.

[0049] As used herein, “nick” refers to a break in one strand of double-stranded DNA.

[0050] As used herein, “non-natural amino acids” means any amino acid other than the standard 20 amino acids encoded by the human genetic code, and each of the following: D-Isomers included: Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val. Non-natural amino acids may have modified or functionalized side chains (e.g., via linker bonding). Examples of non-natural amino acids include, but are not limited to, allo-isoleucine, 2-amino-3-ethylpentanoic acid, aminoisobutyric acid, aminobutyric acid, azetidine, 7-azatryptophan, 6-azidridine, β-cyclobutylalanine, β-methylisoleucine, 4,4-biphenylalanine, cis-hydroxyproline, cyclobutylglycine, cyclohexylglycine, cyclopentylalanine, cyclopentylglycine, 2,6-dimethyltyrosine, 3,3-diphenylalanine, 4-trans-hydroxy-L-proline, 1-naphthylalanine, 2-naphthylalanine, N-methylalanine, 1-methylhistidine, 3-methylhistidine, N-methyltryptophan, pipecolic acid, 4-pyridylalanine, sarcosine, t-butylalanine, or 3-t-butyltyrosine.

[0051] As used herein, “nucleic acid base” means an unmodified nucleic acid base or a modified nucleic acid base. A nucleic acid base is a heterocyclic moiety. As used herein, an “unmodified nucleic acid base” is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). As used herein, a “modified nucleic acid base” is an atomic group other than unmodified A, T, C, U, or G that can pair with at least one other nucleic acid base. “5-methylcytosine” is a modified nucleic acid base. A universal base is a modified nucleic acid base that can pair with any one of the five unmodified nucleic acid bases.

[0052] As used herein, “nucleic acid sequence” means the sequential order of nucleic acid bases in a nucleic acid or oligonucleotide, independent of any sugar or nucleoside bond modifications.

[0053] As used herein, the reference nucleic acid base sequence number "of sequence" or "of sequence" refers only to the nucleic acid base sequence provided by such sequence number, and therefore, unless otherwise indicated, each sugar moiety and each nucleoside bond independently contains a compound that may be modified or unmodified, regardless of the modifications shown in the referenced sequence number.

[0054] As used herein, "nucleoside" means a compound or fragment of a compound comprising a nucleic acid base and a sugar moiety. The nucleic acid base and sugar moiety are either unmodified or modified, independently of each other.

[0055] As used herein, “oligonucleotide” means a chain of linked nucleosides connected via nucleoside-nucleoside bonds, where each nucleoside and nucleoside-nucleoside bond may be independently modified or unmodified. Unless otherwise indicated, an oligonucleotide consists of 8 to 150 linked nucleosides. As used herein, “modified oligonucleotide” means an oligonucleotide in which at least one nucleoside or nucleoside-nucleoside bond is modified. As used herein, “unmodified oligonucleotide” means an oligonucleotide that contains no nucleoside modifications or nucleoside-nucleoside modifications.

[0056] As used herein, “PAM interaction domain” or “PI domain” means a domain of a guide nucleic acid binder that can bind to or associate with one or more PAM sequences in the non-complementary strand of target DNA. The PAM interaction domain may confer specificity to one or more PAM sequences.

[0057] As used herein, “PAM sequence,” “PAM,” or “protospacer flanking motif” refers to 2 to 9 bound nucleosides in the non-complementary strand of target DNA that interact with the guide nucleic acid binder. The PAM sequence may be flanked at the 5' end of the protospacer sequence. The PAM sequence may be flanked at the 3' end of the protospacer sequence.

[0058] As used herein, the peptide sequence of the reference peptide / polypeptide sequence number “peptide sequence of” or “polypeptide sequence of” or “sequence of” means the linear amide-linked amino acid sequence provided in such sequence number, even if a given peptide or polypeptide includes one or more modified side chains linked to another part.

[0059] As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to a subject. Certain such carriers enable the formulation of a pharmaceutical composition into, for example, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges for oral administration by a subject. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile water, distilled water for injection, sterile saline, sterile buffer solution, or sterile artificial cerebrospinal fluid.

[0060] As used herein, “pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of a compound. Pharmacologically acceptable salts retain the desired biological activity of the parent compound and do not exhibit undesirable toxicological effects.

[0061] As used herein, “pharmaceutical composition” means a mixture of substances suitable for administration to a subject. For example, a pharmaceutical composition may include an activator and a sterile aqueous solution. In certain embodiments, the pharmaceutical composition exhibits activity in a free uptake assay in a particular cell line.

[0062] As used herein, “protein recognition region” means a portion of the guide that interacts with the guide nucleic acid binder.

[0063] As used herein, the “protospacer sequence” of the target DNA refers to the reverse complementary strand of the “target sequence” and is found on the non-complementary strand of the target DNA. In certain embodiments, the sequence of the target recognition region of the guide has at least 90%, 95%, 98%, or 99% identity with the protospacer sequence of the target DNA.

[0064] As used herein, “RuvC domain” or “RuvC” means a cation-dependent endonuclease domain of a protein having an active site containing an aspartic acid (D) catalytic residue. In certain embodiments, the active site of the RuvC domain contains catalytic residues of aspartic acid (D) and glutamic acid (E). In certain embodiments, the RuvC domain cleaves the non-complementary strand of target DNA. In certain embodiments, the RuvC domain cleaves both strands of target DNA. The RuvC domain may be formed from discontinuous amino acids; for example, the RuvC domain of SpCas9 is divided into RuvCI, RuvCII, and RuvCIII parts. The RuvC domain may have one or more substitutions that catalytically inactivate it.

[0065] As used herein, "standard-length internucleoside bond" refers to an internucleoside bond having the structure represented by formula Z1, Z2, or Z3. [ka] For each nucleoside bond group in formula Z1, Z2, or Z3, independently, each X 1 These are independently selected from O and S, X 2 O, NR 1 Selected from CH2 and S, X 3 O, NR 1 Selected from CH2 and S, L is absent, NR 1 , N(R 1 )SO2, -N=, O, C1-C6 alkylene, or C1-C6 heteroalkylene, each R 1 is independently selected from H, C1-C6 alkyl, and substituted C1-C6 alkyl, or two R on the same atom 1 together form =O, R 2 is -OH, -SH, C1-C 22 alkyl, substituted C1-C 22 alkyl, C2-C 22 alkenyl, substituted C2-C 22 alkenyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, heteroaryl, substituted heteroaryl, aryl, and substituted aryl, When a group is substituted, the group comprises one or more substituents selected from halo, -OH, -N(R 1 )2, -O-C1-C6 alkyl, C1-C 22 alkyl, C2-C 22 alkenyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl.

[0066] As used herein, "subject" refers to humans, or non-human animals including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, as well as non-human primates including, but not limited to, monkeys and chimpanzees.

[0067] As used herein, “sugar moiety” means an unmodified sugar moiety or a modified sugar moiety. As used herein, “unmodified sugar moiety” means a 2'-OH(H) ribosyl moiety (“unmodified RNA sugar moiety”), as found in RNA, or a 2'-H(H) deoxyribosyl sugar moiety (“unmodified DNA sugar moiety”), as found in DNA. An unmodified sugar moiety has one hydrogen at each of the 1', 3', and 4' positions, one oxygen at the 3' position, and two hydrogens at the 5' position. As used herein, “modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or sugar surrogate.

[0068] As used herein, “sugar surrogate” means a modified sugar moiety having a non-furanosyl moiety that can bond nucleic acid bases to nucleoside bonds. Modified nucleosides containing sugar surrogates can be incorporated at one or more positions within an oligonucleotide, and such oligonucleotides can be hybridized to complementary oligonucleotides or target nucleic acids.

[0069] As used herein, “target recognition region” refers to a region of 12 to 30 bound nucleosides of a guide that is complementary to the “target sequence” within the complementary strand of the target DNA. The target recognition region may be perfectly complementary to the target sequence within the complementary strand of the target DNA.

[0070] As used herein, “target sequence” refers to 12 to 30 bound nucleoside regions of a nucleic acid target that are complementary to the “target recognition region” of the guide. The target sequence is located within the “complementary strand” of the target DNA.

[0071] As used herein, “therapeutic dose” means the amount of a drug or pharmaceutical composition that provides a therapeutic effect to a subject. For example, a therapeutic dose improves the symptoms of a disease.

[0072] As used herein, “to treat” means to improve the disease or condition of interest by administering the agent or compound described herein. In certain embodiments, treating the subject means improving the symptoms compared to the same symptoms in the absence of treatment. In certain embodiments, treatment means reducing the severity or frequency of symptoms, delaying the onset of symptoms, delaying the progression of symptoms, or delaying the severity or frequency of symptoms.

[0073] A certain embodiment This disclosure provides the following non-limiting numbered embodiments.

[0074] Embodiment 1. A polypeptide comprising a region of at least 900, at least 1000, at least 1100, at least 1200, or at least 1300 bound amino acids, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any equal-length portion of SEQ ID NOs. 12, 13, or 20, wherein the polypeptide comprises a RuvC domain and an HNH domain, and optionally, the polypeptide is isolated.

[0075] Embodiment 2. A polypeptide comprising a region, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NOs: 12, 13, or 20, and optionally, the polypeptide is isolated.

[0076] Embodiment 3. A polypeptide wherein the amino acid sequence of the polypeptide is at least 85%, at least 90%, at least 95%, or 100% identical to any of SEQ ID NOs: 12, 13, or 20, and optionally the polypeptide is isolated.

[0077] Embodiment 4. A polypeptide according to Embodiment 1 or 2, comprising the region described above.

[0078] Embodiment 5. The polypeptide according to Embodiment 1 or 2, further comprising heterogeneous domains.

[0079] Embodiment 6. The polypeptide according to Embodiment 5, wherein the heterogeneous domain is selected from a transcription activator, a transcription repressor, a methyltransferase, a demethylase, a deaminase, an acetyltransferase, or a deacetylase.

[0080] Embodiment 7. The polypeptide according to any one of Embodiments 1 to 6, wherein the RuvC domain is comprised of subdomains having amino acid sequences that are at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical with SEQ ID NOs. 85, 92, and 94, or SEQ ID NOs. 97, 104, and 106, or SEQ ID NOs. 109, 116, and 118.

[0081] Embodiment 8. The polypeptide according to any one of Embodiments 1 to 7, wherein the HNH domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of SEQ ID NOs. 93, 105, or 117.

[0082] Embodiment 9. The polypeptide according to any one of Embodiments 1 to 8, wherein the polypeptide comprises an REC lobe.

[0083] Embodiment 10. The polypeptide according to Embodiment 9, wherein the REC lobe has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs. 91, 103, or 115.

[0084] Embodiment 11. The polypeptide according to any one of Embodiments 1 to 10, wherein the polypeptide comprises an NUC lobe.

[0085] Embodiment 12. The polypeptide according to Embodiment 11, wherein the NUC lobe has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 96, 108, or 120.

[0086] Embodiment 13. The polypeptide according to any one of Embodiments 1 to 12, wherein the polypeptide comprises an REC lobe and an NUC lobe.

[0087] Embodiment 14. The polypeptide according to Embodiment 13, wherein the REC lobe has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 91, and the NUC lobe has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 96.

[0088] Embodiment 15. The polypeptide according to Embodiment 14, wherein the REC lobe has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 103, and the NUC lobe has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 108.

[0089] Embodiment 16. The polypeptide according to Embodiment 14, wherein the REC lobe has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115, and the NUC lobe has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 120.

[0090] Embodiment 17. The polypeptide according to any one of Embodiments 1 to 16, wherein the polypeptide has exactly two catalytically active nuclease active sites.

[0091] Embodiment 18. The polypeptide according to any one of Embodiments 1 to 16, wherein the polypeptide has strictly one catalytically active nuclease active site.

[0092] Embodiment 19. The polypeptide according to any one of Embodiments 1 to 17, wherein the polypeptide has 0 catalytically active nuclease active sites.

[0093] Embodiment 20. A polypeptide comprising a region having at least 800, at least 900, at least 1000, or at least 1050 bound amino acids, wherein the amino acid sequence has at least 85%, at least 90%, at least 95%, or 100% identity with any equal-length portion of SEQ ID NOs. 241, 242, 245, 247, 314, or 323, and optionally, the polypeptide is isolated.

[0094] Embodiment 21. A polypeptide comprising a region having at least 900, at least 1000, at least 1100, or at least 1200 bound amino acids, wherein the amino acid sequence has at least 85%, at least 90%, at least 95%, or 100% identity with the isolength portion of SEQ ID NO: 260, and optionally, the polypeptide is isolated.

[0095] Embodiment 22. A polypeptide comprising a region having at least 900, at least 1000, at least 1100, at least 1200, or at least 1300 linked amino acids, wherein the amino acid sequence has at least 85%, at least 90%, at least 95%, or 100% identity with any equal-length portion of SEQ ID NOs. 266-268, 271, 274-275, 279, 282-284, 288-289, 292, 297, or 299-302, and optionally, the polypeptide is isolated.

[0096] Embodiment 23. A polypeptide comprising a region, wherein the region has an amino acid sequence having at least 85%, at least 90%, at least 95%, or 100% identity with any equal-length portion of SEQ ID NOs: 241, 242, 245, 247, 260, 266-268, 271, 274-275, 279, 282-284, 288-289, 292, 297, 299-302, 314, or 323, and optionally, the polypeptide is isolated.

[0097] Embodiment 24. A polypeptide wherein the amino acid sequence of the polypeptide is at least 85%, at least 90%, at least 95%, or 100% identical to any of SEQ ID NOs: 241, 242, 245, 247, 260, 266-268, 271, 274-275, 279, 282-284, 288-289, 292, 297, 299-302, 314, or 323, and optionally the polypeptide is isolated.

[0098] Embodiment 25. A guide comprising a protein recognition element, wherein the protein recognition element is bound to a polypeptide according to any one of Embodiments 1 to 24.

[0099] Embodiment 26. The guide according to Embodiment 25, wherein the guide is a dual guide consisting of two oligonucleotides linked by a double-stranded region.

[0100] Embodiment 27. The guide according to Embodiment 25, wherein the guide is a single guide comprising an oligonucleotide, and the protein recognition element is the protein recognition region of the oligonucleotide.

[0101] Embodiment 28. The guide according to Embodiment 27, wherein the nucleic acid base sequence of the protein recognition region of the guide has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 129, 130, 131, or 139.

[0102] Embodiment 29. The guide according to Embodiment 27, wherein the nucleic acid base sequence of the protein recognition region of the guide has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 520-521, 524, 526, 539, 545-547, 550, 553-554, 558, 561-563, 567, 568, 571, 576, 578-581, 593, or 602.

[0103] Embodiment 30. The guide according to any one of Embodiments 26 to 29, wherein the guide comprises a modified oligonucleotide.

[0104] Embodiment 31. The guide according to any one of Embodiments 26 to 30, wherein the guide comprises at least one modified sugar moiety.

[0105] Embodiment 32. The guide according to Embodiment 31, wherein the modified sugar portion is selected from 2'-OMe and 2'-F.

[0106] Embodiment 33. The guide according to Embodiment 32, wherein the guide contains a 2'-OMe or 2'-F sugar moiety within the first five nucleosides at the 5' end of the guide or within the last five nucleosides at the 3' end.

[0107] Embodiment 34. The guide according to any one of Embodiments 26 to 33, wherein the guide includes a modified nucleoside bond.

[0108] Embodiment 35. The guide according to Embodiment 34, wherein the modified nucleoside bond is a phosphorothioate nucleoside bond.

[0109] Embodiment 36. The guide according to Embodiment 34 or 35, wherein the guide includes at least one modified internucleoside bond within the first five nucleosides at the 5' end of the guide or within the last five nucleosides at the 3' end.

[0110] Embodiment 37. The guide according to any one of Embodiments 27 to 36, wherein the guide is made of an oligonucleotide.

[0111] Embodiment 38. The guide according to any one of Embodiments 25 to 37, wherein the guide includes a DNA recognition element that is at least 90%, at least 95%, or 100% complementary to the target sequence.

[0112] Embodiment 39. An editing system comprising a polypeptide according to any one of Embodiments 1 to 24 and a guide according to any one of Embodiments 25 to 38.

[0113] Embodiment 40. An editing system, a. A nucleic acid encoding a polypeptide according to any one of Embodiments 1 to 24, b. An editing system comprising the guide described in any one of embodiments 25 to 38.

[0114] Embodiment 41. An editing system, a. A nucleic acid encoding a polypeptide according to any one of Embodiments 1 to 24, b. An editing system comprising a nucleic acid encoding a guide as described in any one of embodiments 25 to 29.

[0115] Embodiment 42. The editing system according to Embodiment 40 or 41, wherein the nucleic acid encoding the polypeptide is exogenous mRNA.

[0116] Embodiment 43. The editing system according to Embodiment 40 or 41, wherein the nucleic acid encoding the polypeptide is DNA.

[0117] Embodiment 44. The editing system according to any one of Embodiments 41 to 43, wherein the nucleic acid encoding the guide is exogenous mRNA.

[0118] Embodiment 45. The editing system according to any one of Embodiments 41 to 43, wherein the nucleic acid encoding the guide is DNA.

[0119] Embodiment 46. A composition comprising the editing system described in any one of Embodiments 39 to 45 and an LNP.

[0120] Embodiment 47. A viral vector comprising the editing system described in Embodiment 41.

[0121] Embodiment 48. A method for editing a target nucleic acid, comprising administering the composition described in Embodiment 46 or the viral vector described in Embodiment 47 to a target.

[0122] Embodiment 49. A method for forming a double-strand break or nick in a target nucleic acid, comprising administering the composition described in Embodiment 46 or the viral vector described in Embodiment 47 to the target.

[0123] Embodiment 50. A method for editing a target nucleic acid, comprising contacting a cell with the composition described in Embodiment 46 or the viral vector described in Embodiment 47.

[0124] Embodiment 51. A method for forming double-strand breaks or nicks in a target nucleic acid, comprising contacting a cell with the composition described in Embodiment 46 or the viral vector described in Embodiment 47.

[0125] Embodiment 52. A composition of Embodiment 45 or a viral vector of Embodiment 46 for use in therapy.

[0126] Embodiment 53. A polypeptide comprising a region having at least 800, at least 900, at least 1000, or at least 1050 bound amino acids, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity with any equal-length portion of SEQ ID NOs. 4-23 or 231-323, and optionally, the polypeptide is isolated.

[0127] Embodiment 54. A polypeptide comprising a region having at least 900, at least 1000, or at least 1100 linked amino acids, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity with the isolength portions of SEQ ID NOs. 6-23, 250-308, and optionally, the polypeptide is isolated.

[0128] Embodiment 55. A polypeptide comprising a region having at least 900, at least 1000, at least 1100, at least 1200, or at least 1300 bound amino acids, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity with any equal-length portion of SEQ ID NOs. 10-23 or 266-308, and optionally, the polypeptide is isolated.

[0129] Embodiment 56. A polypeptide comprising a region, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity with any of SEQ ID NOs: 4-23 or 231-323, and optionally, the polypeptide is isolated.

[0130] Embodiment 57. A polypeptide wherein the amino acid sequence of the polypeptide has at least 85%, at least 90%, at least 95%, or 100% identity with any of SEQ ID NOs: 4-23 or 231-323, and optionally the polypeptide is isolated.

[0131] Embodiment 58. The polypeptide according to Embodiment 57, wherein the amino acid sequence of the polypeptide is at least 85%, at least 90%, at least 95%, or 100% identical to any of SEQ ID NOs: 12, 13, 20, 241, 242, 245, 247, 260, 266, 267, 268, 271, 274, 275, 279, 282, 283, 284, 288, 289, 292, 297, 299, 300, 301, 302, 314, or 323.

[0132] Embodiment 59. A polypeptide according to any one of Embodiments 53 to 56, comprising the region described above.

[0133] Embodiment 60. A polypeptide according to any one of Embodiments 53 to 56, comprising heterogeneous domains.

[0134] Embodiment 61. The polypeptide according to Embodiment 60, wherein the heterogeneous domain is selected from a transcription activator, a transcription repressor, a methyltransferase, a demethylase, a deaminase, an acetyltransferase, or a deacetylase.

[0135] Embodiment 62. The polypeptide according to any one of Embodiments 53 to 56 or 59 to 61, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity with any equal-length portion of SEQ ID NOs: 13, 241, 267, 275, 284, 289, or 314.

[0136] Embodiment 63. The polypeptide according to any one of Embodiments 53 to 62, wherein the polypeptide comprises a non-PI region and a PI domain, and the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 3186, 3188, 3190, 3192, 3194, 3196, or 3198.

[0137] Embodiment 64. The polypeptide according to any one of Embodiments 53 to 62, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3186, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3187.

[0138] Embodiment 65. The polypeptide according to any one of Embodiments 53 to 62, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3188, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3189.

[0139] Embodiment 66. The polypeptide according to any one of Embodiments 53 to 62, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3190, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3191.

[0140] Embodiment 67. The polypeptide according to any one of Embodiments 53 to 62, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3192, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3193.

[0141] Embodiment 68. The polypeptide according to any one of Embodiments 53 to 62, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3194, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3195.

[0142] Embodiment 69. The polypeptide according to any one of Embodiments 53 to 62, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3196, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3197.

[0143] Embodiment 70. The polypeptide according to any one of Embodiments 53 to 62, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3198, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3199.

[0144] Embodiment 71. The polypeptide according to any one of Embodiments 53 to 70, wherein the polypeptide has exactly two catalytically active nuclease active sites.

[0145] Embodiment 72. The polypeptide according to any one of Embodiments 53 to 70, wherein the polypeptide has strictly one catalytically active nuclease active site.

[0146] Embodiment 73. The polypeptide according to any one of Embodiments 53 to 70, wherein the polypeptide has 0 catalytically active nuclease active sites.

[0147] Embodiment 74. A polypeptide according to any one of Embodiments 53 to 73, comprising a nuclear localization signal.

[0148] Embodiment 75. The polypeptide according to Embodiment 74, wherein the nuclear localization signal is located at the N-terminus of the polypeptide.

[0149] Embodiment 76. The polypeptide according to Embodiment 74, wherein the nuclear localization signal is located at the C-terminus of the polypeptide.

[0150] Embodiment 77. The polypeptide according to Embodiment 74, wherein the polypeptide comprises one and two nuclear localization signals at the N-terminus and C-terminus, respectively.

[0151] Embodiment 78. A nucleic acid encoding a polypeptide according to any one of Embodiments 53 to 77.

[0152] Embodiment 79. The nucleic acid according to Embodiment 78, wherein the nucleic acid is DNA.

[0153] Embodiment 80. The nucleic acid according to Embodiment 78, wherein the nucleic acid is mRNA containing a coding region encoding a polypeptide described in any one of Embodiments 53 to 77.

[0154] Embodiment 81. The nucleic acid according to Embodiment 78, wherein the mRNA is an exogenous mRNA containing a coding region encoding a polypeptide described in any one of Embodiments 53 to 73.

[0155] Embodiment 82. mRNA according to Embodiment 80 or 81, comprising a 5' cap structure.

[0156] Embodiment 83. mRNA according to any one of Embodiments 80 to 82, comprising a 3'-poly(A) tail.

[0157] Embodiment 84.5'-UTR, comprising the mRNA according to any one of Embodiments 80 to 83.

[0158] mRNA according to any one of embodiments 80 to 84, including embodiment 85.3'-UTR.

[0159] Embodiment 86. mRNA according to Embodiment 80 or 81, comprising a 5'-cap structure and a 3'-poly(A) tail.

[0160] Embodiment 87. The mRNA according to any one of Embodiments 80 to 86, wherein the coding region is codon-optimized for expression in eukaryotic cells.

[0161] Embodiment 88. The mRNA according to Embodiment 87, wherein the coding region is codon-optimized for expression in mammalian cells.

[0162] Embodiment 89. The mRNA according to Embodiment 88, wherein the mammalian cell is a human cell.

[0163] Embodiment 90. The mRNA according to any one of Embodiments 80 to 89, wherein each nucleic acid base of the mRNA is selected from adenine, guanine, cytosine, uracil, thymine, and N1-methylpseudolacil.

[0164] Embodiment 91. The mRNA according to any one of Embodiments 80 to 89, wherein each nucleic acid base of the mRNA is selected from adenine, guanine, cytosine, and N1-methylpseudracil.

[0165] Embodiment 92. The mRNA according to any one of Embodiments 80 to 89, wherein each nucleic acid base of the mRNA is selected from adenine, guanine, cytosine, and uracil.

[0166] Embodiment 93. A guide comprising a protein recognition region, wherein the protein recognition region interacts with a polypeptide as described in any one of Embodiments 53 to 57.

[0167] Embodiment 94. The guide according to Embodiment 93, wherein the guide is a dual guide.

[0168] Embodiment 95. The guide according to Embodiment 93, wherein the guide is a single guide.

[0169] Embodiment 96. A guide, the following formula: T1-DXS 1a -B1-S 1b -H1-S 1b '-B2-S 1a '-L1-S2-H2-S2'-L2-S3-H3-S3'-(L3-S4-H4-S4') n -(L4-S5-H5-S5') m -T2 It consists of oligonucleotides that follow the formula, In the array, D consists of 17 to 25 linked nucleosides, and the nucleic acid base sequence of D is complementary to the nucleic acid base sequence of the target DNA. X consists of one or two bound nucleosides that are absent or not complementary to the sequence of the target DNA. Each of T1 and T2 is either absent or independently consists of 1 to 30 bound nucleosides. S 1a and S 1aEach of these consists of 6 to 10 bonded nucleosides, S 1a The nucleic acid base sequence is S 1a It is 100% complementary to the nucleic acid base sequence of ', S 1b and S 1b Each of these consists of 2 to 14 bonded nucleosides, S 1b The nucleic acid base sequence is S 1b It is 100% complementary to the nucleic acid base sequence of ', S2 and S2' each consist of 2 to 6 bonded nucleosides, and the nucleic acid base sequence of S2 is 100% complementary to the nucleic acid base sequence of S2'. S3 and S3' each consist of 2 to 12 bonded nucleosides, and the nucleic acid base sequence of S3 is 100% complementary to the nucleic acid base sequence of S3'. S4 and S4' each consist of 4 to 14 bonded nucleosides, and the nucleic acid base sequence of S4 is 100% complementary to the nucleic acid base sequence of S4'. S5 and S5' each consist of 4 to 11 bonded nucleosides, and the nucleic acid base sequence of S5 is 100% complementary to the nucleic acid base sequence of S5'. n is 0 or 1, m is 0 or 1, B1 is either absent or consists of 1 to 3 bonded nucleosides. B2 is either absent or consists of 1 to 4 bonded nucleosides. Except when both B1 and B2 are absent, B1 is not equal to B2. Each of H1, H2, H3, H4, and H5 independently consists of 3 to 6 bonded nucleosides. L1 consists of 2-3 bonded nucleosides, L2, L3, and L4 are guides, each consisting of 0 to 10 bound nucleosides.

[0170] Embodiment 97. The guide according to Embodiment 96, wherein the guide includes a protein recognition region that binds to a polypeptide described in any one of Embodiments 53 to 77.

[0171] Embodiment 98.T1 is absent from the guide described in Embodiment 96 or 97.

[0172] The guide described in any one of embodiments 96 to 98, wherein embodiment 99.T2 is absent.

[0173] Embodiment 100.T2 is a guide according to any one of Embodiments 96 to 99, wherein T2 consists of 2 to 6 bonded nucleosides containing uracil nucleic acid bases.

[0174] The guide described in any one of Embodiments 96 to 100, in which Embodiment 101.X is absent.

[0175] Embodiment 102.n, the guide according to any one of Embodiments 96 to 101, wherein n is 1 and m is 0.

[0176] The guide according to any one of embodiments 96 to 102, wherein both n and m are 1.

[0177] The guide according to any one of Embodiments 96 to 103, wherein the number of nucleosides in Embodiment 104.B2 is greater than the number of nucleosides in B1.

[0178] Embodiment 105. The guide according to any one of Embodiments 96 to 104, wherein each L1 nucleoside is adenosine.

[0179] Embodiment 106. The guide according to any one of Embodiments 96 to 105, wherein the guide is bound to the polypeptide described in any one of Embodiments 53 to 77.

[0180] Embodiment 107. T1 and X are absent. T2 is either absent or consists of 2-6 bound uridine molecules. S 1a and S 1a Each of these consists of seven bonded nucleosides. S 1b and S 1b Each of these consists of 3 to 9 bonded nucleosides. S2 and S2' each consist of three bonded nucleosides. S3 and S3' each consist of four bonded nucleosides. S4 and S4' each consist of five bonded nucleosides. If S5 and S5' are present, each consists of 7 to 9 bonded nucleosides. n is 1, m is 0 or 1, B1 consists of one nucleoside, B2 consists of four bonded nucleosides, If H1, H2, H3, and H4 are present, each consists of four bonded nucleosides. L1 consists of two bound adenosine nucleosides, L2 consists of five bonded nucleosides, L3 consists of 0 nucleosides, The guide according to embodiment 96 or 97, wherein L4, if present, consists of four bound nucleosides.

[0181] Embodiment 108.S 1b and S 1b The guide according to Embodiment 107, wherein each of the seven bonded nucleosides is represented.

[0182] Embodiment 109. The guide according to Embodiment 107 or 108, wherein S5 and S5' each consist of seven bonded nucleosides.

[0183] The guide according to any one of Embodiments 107 to 109, wherein the nucleic acid base sequence of Embodiment 110.B2 is AAAG.

[0184] The guide according to Embodiment 107 or 108, wherein the nucleic acid base sequence of Embodiment 111.L2 is AAACU.

[0185] Embodiment 112. The guide according to any one of Embodiments 107 to 111, wherein the nucleic acid base sequence of L3 is UUUUAA.

[0186] Embodiment 113. The guide according to any one of Embodiments 107 to 112, wherein the nucleic acid base sequence of H2 is GUCA.

[0187] Embodiment 114.n, the guide according to any one of Embodiments 107 to 113, wherein n is 1 and m is 0.

[0188] Embodiment 115. The guide according to any one of Embodiments 107 to 114, wherein both n and m are 1.

[0189] Embodiment 116.S 1b and S 1b The guide according to Embodiment 107, wherein each of the ' nucleosides consists of seven bonded nucleosides, n is 1, m is 0, the nucleic acid base sequence of B2 is AAAG, the nucleic acid base sequence of L2 is AAACU, the nucleic acid base sequence of L3 is UUUUAA, and the nucleic acid base sequence of H2 is GUCA.

[0190] Embodiment 117. The guide according to any one of Embodiments 107 to 116, wherein the guide comprises at least 40, at least 50, at least 60, or at least 70 nucleoside regions, and the nucleic acid base sequences of the regions have at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any isolength region of SEQ ID NOs. 131, 961-964, 1016-1020, 1052-1070, 1313-1314, or 2825.

[0191] Embodiment 118. The guide according to Embodiment 117, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NOs. 131, 961-964, 1016-1020, 1052-1070, 1313-1314, or 2825.

[0192] Embodiment 119.D is a guide according to any one of Embodiments 107 to 118, comprising 20 to 23 nucleosides having a nucleic acid base sequence that is 100% complementary to the nucleic acid base sequence of the target DNA.

[0193] Embodiment 120. T1 and X are absent. T2 is either absent or consists of six bound uridines. S 1a and S 1a Each of these consists of nine bonded nucleosides. S 1b and S 1b Each of these consists of 2 to 8 ink-coated nucleosides. S2 and S2' each consist of six bonded nucleosides. S3 and S3' each consist of seven bonded nucleosides. n and m are 0, B1 consists of three nucleosides, B2 consists of four bonded nucleosides, H1 consists of four bonded nucleosides, H2 consists of five bonded nucleosides, H3 consists of four bonded nucleosides, L1 consists of two bound adenosine nucleosides, The guide according to embodiment 96 or 97, wherein L2 consists of zero bound nucleosides.

[0194] Embodiment 121.S 1b and S 1b' each consist of 4 linked nucleosides, the guide of embodiment 120.

[0195] Embodiment 122. The guide of embodiment 120 or 121, wherein L2 consists of 0 linked nucleosides, and T2 consists of 6 linked uridines.

[0196] Embodiment 123. The guide of any one of embodiments 120 to 122, wherein the nucleobase sequence of B2 is UAAC.

[0197] Embodiment 124. The guide of any one of embodiments 120 to 123, wherein the nucleobase sequence of L2 is GAACUC.

[0198] Embodiment 125. The guide of any one of embodiments 120 to 124, wherein the nucleobase sequence of H2 is UUUAU.

[0199] Embodiment 126. S 1b and S 1b ' each consist of 4 linked nucleosides, n and m are 0, the nucleobase sequence of B2 is UACC, the nucleobase sequence of L2 is GAACUC, and the nucleobase sequence of H2 is UUUAU, the guide of embodiment 120.

[0200] Embodiment 127. The guide of any one of embodiments 120 to 126, wherein said guide comprises a region having a nucleobase sequence with at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NO: 520, 965 to 968, 1071 to 1094, or 1315 to 1318, and wherein said region comprises at least 40, at least 50, at least 60, or at least 70 nucleosides.

[0201] Embodiment 128. The guide according to Embodiment 127, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NOs. 520, 965-968, 1071-1094, or 1315-1318.

[0202] Embodiment 129.D is a guide according to any one of Embodiments 120 to 127, comprising 20 to 23 nucleosides having a nucleic acid base sequence that is 100% complementary to the nucleic acid base sequence of the target DNA.

[0203] Embodiment 130. T1 and X are absent. T2 is either absent or consists of 1 to 7 bonded nucleosides. S 1a and S 1a Each of these consists of seven bonded nucleosides. S 1b and S 1b Each of these consists of 4 to 12 bonded nucleosides. S2 and S2' each consist of five bonded nucleosides. S3 and S3' each consist of six bonded nucleosides. S4 and S4' each consist of six bonded nucleosides. If S5 and S5' are present, each consists of seven bonded nucleosides. n is 1, m is 0 or 1, B1 consists of one nucleoside, B2 consists of three bonded nucleosides, Each of H1, H2, and H3 consists of four bonded nucleosides. If H4 and H5 are present, they consist of three bonded nucleosides. L1 consists of three linked adenosine nucleosides, L2 and L3 consist of 0 bound nucleosides. The guide according to embodiment 96 or 97, wherein L4, if present, consists of 1 to 7 bound nucleosides.

[0204] Embodiment 131.S 1b and S 1b The guide according to Embodiment 130, wherein each of the six bonded nucleosides is represented.

[0205] The guide according to any one of Embodiments 130 to 131, wherein the nucleic acid base sequence of Embodiment 132.B2 is GAG.

[0206] Embodiment 133. The guide according to any one of Embodiments 130 to 132, wherein the nucleic acid base sequence of H2 is AUCC.

[0207] Embodiment 134.S 1b and S 1b The guide according to Embodiment 130, wherein each of the ' nucleosides consists of six bonded nucleosides, n is 1, m is 0, the nucleic acid base sequence of B2 is GAG, and the nucleic acid base sequence of H2 is AUCC.

[0208] Embodiment 135. The guide according to any one of Embodiments 130 to 134, wherein the guide includes a region having a nucleic acid sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of SEQ ID NOs. 546, 973-976, 1021-1023, 1095-1126, or 1319-1342, and the region includes at least 40, at least 50, at least 60, or at least 70 nucleosides.

[0209] Embodiment 136. The guide according to Embodiment 135, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NOs. 546, 973-976, 1021-1023, 1095-1126, or 1319-1342.

[0210] Embodiment 137. The guide according to any one of Embodiments 130 to 136, wherein D consists of 20 to 24 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of the target DNA.

[0211] Embodiment 138. T1 and X are absent, T2 is absent or consists of 1 to 6 linked nucleosides, S 1a and S 1a ' each consist of 7 linked nucleosides, S 1b and S 1b ' each consist of 4 to 12 modified nucleosides, S2 and S2' each consist of 4 linked nucleosides, S3 and S3' each consist of 4 linked nucleosides, S4 and S4' each consist of 5 linked nucleosides, S5 and S5', when present, each consist of 7 linked nucleosides, n is 1, m is 0 or 1, B1 consists of 1 nucleoside, B2 consists of 3 nucleosides, H1 consists of 4 linked nucleosides, H2 consists of 3 linked nucleosides, H3 consists of 6 linked nucleosides, H4 consists of 4 linked nucleosides, H5, when present, consists of 3 linked nucleosides, L1 consists of 2 linked adenosine nucleosides, L2 consists of 2 linked nucleosides, L3 consists of 0 linked nucleosides, The guide according to embodiment 96 or 97, wherein L4, if present, consists of seven bound nucleosides.

[0212] Embodiment 139.S 1b and S 1b The guide according to Embodiment 138, wherein each of the ' ' consists of 10 bonded nucleosides.

[0213] Embodiment 140.n is the guide described in Embodiment 138 or 139, where m is 1 and m is 0.

[0214] The guide according to any one of Embodiments 138 to 140, wherein the nucleic acid base sequence of Embodiment 141.B2 is GAG.

[0215] Embodiment 142. The guide according to any one of Embodiments 138 to 141, wherein the nucleic acid base sequence of L2 is AA.

[0216] Embodiment 143. The guide according to any one of Embodiments 138 to 142, wherein the nucleic acid base sequence of H2 is UAA.

[0217] Embodiment 144.S 1b and S 1b The guide according to Embodiment 138, wherein each of the ' consists of 10 bonded nucleosides, n is 1, m is 0, the nucleic acid base sequence of B2 is GAG, the nucleic acid base sequence of L2 is AA, and the nucleic acid base sequence of H2 is UAA.

[0218] Embodiment 145. The guide according to any one of Embodiments 138 to 144, wherein the guide includes a region having a nucleic acid sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of Sequence IDs 562, 985-989, 1027-1029, 1153-1182, or 1362-1390, and the region includes at least 40, at least 50, at least 60, or at least 70 nucleosides.

[0219] Embodiment 146. The guide according to Embodiment 145, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NOs. 562, 985-989, 1027-1029, 1153-1182, or 1362-1390.

[0220] Embodiment 147.D is a guide according to any one of Embodiments 138 to 146, comprising 20 to 24 nucleosides having nucleic acid base sequences that are 100% complementary to the nucleic acid base sequence of the target DNA.

[0221] Embodiment 148. T1 and X are absent. T2 is either absent or consists of 2-5 bonded nucleosides. S 1a and S 1a Each of these consists of nine bonded nucleosides. S 1b and S 1b Each of these consists of 2 to 10 bonded nucleosides. S2 and S2' each consist of four bonded nucleosides. S3 and S3' each consist of nine bonded nucleosides. n and m are 0, B1 consists of one nucleoside, B1 consists of three nucleosides, H1 consists of four bonded nucleosides, H2 consists of three bonded nucleosides, H3 consists of four bonded nucleosides, L1 consists of two bound adenosine nucleosides, The guide according to embodiment 96 or 97, wherein L2 consists of seven bonded nucleosides.

[0222] Embodiment 149.S 1b and S 1bThe guide according to Embodiment 148, wherein each of the six bonded nucleosides is represented.

[0223] The guide according to Embodiment 148 or 149, wherein the nucleic acid base sequence of Embodiment 150.B2 is CUA.

[0224] Embodiment 151. The guide according to any one of Embodiments 148 to 150, wherein the nucleic acid base sequence of L2 is GUGUUUA.

[0225] Embodiment 152. The guide according to any one of Embodiments 148 to 151, wherein the nucleic acid base sequence of H2 is AAA.

[0226] Embodiment 153.S 1b and S 1b The guide according to Embodiment 148, wherein each of the ' consists of 10 bonded nucleosides, n and m are 0, the nucleic acid base sequence of B2 is CUA, the nucleic acid base sequence of L2 is GUGUUUA, and the nucleic acid base sequence of H2 is AAA.

[0227] Embodiment 154. The guide according to any one of Embodiments 148 to 153, wherein the guide includes a region having a nucleic acid sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of SEQ ID NOs: 593, 1006-1010, 1272-1292, 1394-1408, or 2827-2833, and the region includes at least 40, at least 50, at least 60, or at least 70 nucleosides.

[0228] Embodiment 155. The guide according to Embodiment 160, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NOs. 593, 1006-1010, 1272-1292, 1394-1408, and 2827-2833.

[0229] Embodiment 156.D is a guide according to any one of Embodiments 148 to 155, comprising 20 to 24 nucleosides having nucleic acid base sequences that are 100% complementary to the nucleic acid base sequence of the target DNA.

[0230] Embodiment 157. T1 and X are absent. T2 is either absent or consists of three bonded nucleosides. S 1a and S 1b But together, as well as S 1a 'and S 1b 'These combine to form a structure consisting of 12 to 20 bonded nucleosides, S2 and S2' each consist of three bonded nucleosides. S3 and S3' each consist of three bonded nucleosides. S3 and S3' each consist of 10 to 13 bonded nucleosides. n is 1 and m is 0. B1 consists of 0 nucleosides, B2 consists of 0 nucleosides, H 1、 H2, H3, and H4 consist of four bonded nucleosides. L1 consists of two bound adenosine nucleosides, L2 consists of two bonded nucleosides, The guide according to embodiment 96 or 97, wherein L3 consists of six bound nucleosides.

[0231] Embodiment 158.S 1a and S 1b But together, as well as S 1a 'and S 1b The guide according to Embodiment 157, wherein together, each consists of 15 bonded nucleosides.

[0232] Embodiment 159. The guide according to Embodiment 157 or 158, wherein L3 consists of three bonded nucleosides.

[0233] Embodiment 160. A guide according to any one of Embodiments 157 to 159, wherein S3 and S3' each consist of 13 bonded nucleosides.

[0234] Embodiment 161. The guide according to any one of Embodiments 157 to 159, wherein the nucleic acid base sequence of L2 is GU.

[0235] Embodiment 162. The guide according to Embodiment 160 or 161, wherein the nucleic acid base sequence of H2 is GAAA.

[0236] Embodiment 163.S 1a and S 1b But together, as well as S 1a 'and S 1b The guide according to Embodiment 157, wherein each of the following is composed of 15 bonded nucleosides, n is 1, m is 0, the nucleic acid base sequence of L2 is GU, and the nucleic acid base sequence of H2 is GAAA.

[0237] Embodiment 164. The guide according to any one of Embodiments 157 to 163, wherein the guide includes a region having a nucleic acid sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of SEQ ID NOs. 568, 990-993, 1030-1034, 1183-1206, or 1391-1393, and the region includes at least 40, at least 50, at least 60, or at least 70 nucleosides.

[0238] Embodiment 165. The guide according to Embodiment 164, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NOs. 568, 990-993, 1030-1034, 1183-1206, or 1391-1393.

[0239] Embodiment 166. The guide according to any one of Embodiments 158 to 165, which consists of 20 to 24 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of the target DNA.

[0240] Embodiment 167. A guide, which is represented by the following formula: T1-D-X-S 1a -B1-S 1b -H1-S 1b '-B2-S 1a '-L1-S2-H2-S2'-L2-S3-H3-S3'-(L3-S4-H4-S4') n -T2 consists of an oligonucleotide conforming to the formula, wherein in the sequence, D consists of 17 to 25 linked nucleosides, and the nucleobase sequence of D is complementary to the nucleobase sequence of the target DNA, X is absent, or consists of 1 to 2 linked nucleosides that are not complementary to the nucleobase sequence of the target DNA, each of T1 and T2 is independently absent or consists of 1 to 30 linked nucleosides, S 1a and S 1a ' each consist of 7 linked nucleosides, and the nucleobase sequence of S 1a is 100% complementary to the nucleobase sequence of S 1a ', S 1b and S 1b ' each consist of 4 to 12 linked nucleosides, and the nucleobase sequence of S 1b is 100% complementary to the nucleobase sequence of S 1b ', S2 and S2' each consist of 6 linked nucleosides, and the nucleobase sequence of S2 is 100% complementary to the nucleobase sequence of S2', S3 and S3' each consist of 5 or 6 linked nucleosides, and the nucleobase sequence of S3 is 100% complementary to the nucleobase sequence of S3', If S4 and S4' are present, each consists of six bound nucleosides, and the nucleic acid base sequence of S4 is 100% complementary to the nucleic acid base sequence of S4'. n is 0 or 1, B1 consists of one nucleoside, B2 consists of three bonded nucleosides, Each of H1, H2, H3, and H4 independently consists of 3 to 4 bonded nucleosides. L1 consists of 17 bonded nucleosides, L2 consists of 0 bonded nucleosides, L3 is a guide consisting of eight bonded nucleosides.

[0241] Embodiment 168.S 1b and S 1b The guide according to Embodiment 167, wherein each of the eight bonded nucleosides is represented.

[0242] The guide according to Embodiment 167 or 168, wherein the nucleic acid base sequence of Embodiment 169.B2 is GAA.

[0243] The guide according to any one of Embodiments 167 to 169, wherein the nucleic acid base sequence of Embodiment 170.L2 is AAAAAUUUAUUCAAAAC (Sequence ID 78).

[0244] Embodiment 171. The guide according to any one of Embodiments 167 to 170, wherein the nucleic acid base sequence of H2 is GAAA.

[0245] Embodiment 172.S 1b and S 1b The guide according to Embodiment 171, wherein each of the ' nucleotides consists of eight bonded nucleosides, n and m are 0, the nucleic acid base sequence of B2 is GAA, the nucleic acid base sequence of L2 is AAAAAUUUAUUCAAAAC (SEQ ID NO: 78), and the nucleic acid base sequence of H2 is GAAA.

[0246] Embodiment 173. The guide according to any one of Embodiments 167 to 172, wherein the guide includes a region having a nucleic acid sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of SEQ ID NOs. 554, 981-984, 1024-1026, 1127-1152, or 1343-1361, and the region includes at least 40, at least 50, at least 60, or at least 70 nucleosides.

[0247] Embodiment 174. The guide according to Embodiment 173, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NOs. 554, 981-984, 1024-1026, 1127-1152, or 1343-1361.

[0248] Embodiment 175.D is a guide according to any one of Embodiments 167 to 174, comprising 20 to 24 nucleosides having nucleic acid base sequences that are 100% complementary to the nucleic acid base sequence of the target DNA.

[0249] Embodiment 176. The guide according to any one of Embodiments 93 to 95, wherein the guide includes a region having a nucleic acid base sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of SEQ ID NOs. 121 to 141, 510 to 602, 961 to 1430, or 1436 to 2833, and the region includes at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 nucleosides.

[0250] Embodiment 177. The guide according to any one of Embodiments 93 to 95, wherein the guide includes a protein recognition region having a nucleic acid base sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of SEQ ID NOs. 121 to 141, 510 to 602, 961 to 1430, or 1436 to 2833.

[0251] Embodiment 178. The guide according to any one of Embodiments 93 to 177, wherein the guide comprises a modified oligonucleotide.

[0252] Embodiment 179. The guide according to any one of Embodiments 93 to 178, wherein the guide comprises at least one modified sugar moiety.

[0253] Embodiment 180. The guide according to Embodiment 179, wherein the modified sugar moiety is selected from 2'-OMe and 2'-F.

[0254] Embodiment 181. The guide according to Embodiment 179, wherein the modified oligonucleotide contains a 2'-OMe or 2'-F sugar moiety within the first five nucleosides at the 5' end of the guide or within the last five nucleosides at the 3' end of the guide.

[0255] Embodiment 182. The guide according to Embodiments 179 to 181, wherein the guide includes two to three 2'-OMe sugar moieties at its 5' end and two to three 2'-OMe sugar moieties at its 3' end.

[0256] Embodiment 183. The guide according to Embodiment 182, wherein each non-2'-OMe sugar moiety of the guide contains an unmodified RNA sugar moiety.

[0257] Embodiment 184. The guide according to any one of Embodiments 179 to 183, wherein the guide includes a modified nucleoside bond.

[0258] Embodiment 185. The guide according to Embodiment 184, wherein the modified nucleoside bond is a phosphorothioate nucleoside bond.

[0259] Embodiment 186. The guide according to Embodiment 184 or 185, wherein the guide includes at least one modified internucleoside bond within the first five nucleosides at the 5' end of the guide or within the last five nucleosides at the 3' end of the guide.

[0260] Embodiment 187. The guide according to Embodiment 186, wherein the guide includes 2 to 3 phosphorothioate nucleoside bonds at its 5' end and 2 to 3 phosphorothioate nucleoside bonds at its 3' end.

[0261] Embodiment 188. The guide according to Embodiment 187, wherein each remaining nucleoside bond is an unmodified phosphodiester nucleoside bond.

[0262] Embodiment 189. The guide according to any one of Embodiments 184 to 188, wherein each nucleoside bond in the guide is a standard-length nucleoside bond.

[0263] Embodiment 190. The guide according to any one of Embodiments 93 to 177, wherein the guide comprises an unmodified oligonucleotide.

[0264] Embodiment 191. The guide according to any one of Embodiments 93 to 190, wherein the guide includes a target recognition region that is at least 90%, at least 95%, or 100% complementary to the target DNA sequence.

[0265] Embodiment 192. The guide according to Embodiment 191, wherein the target recognition region comprises 17-25, 19-24, 20-24, 20-23, 20-22, 21-22, 21-23, 21-24, 20, 21, 22, 23, or 24 bound nucleosides.

[0266] Embodiment 193. An editing system comprising a polypeptide according to any one of Embodiments 53 to 77 and a guide according to any one of Embodiments 93 to 192.

[0267] Embodiment 194. An editing system, A nucleic acid according to any one of embodiments 78 to 92, An editing system comprising the guide described in any one of embodiments 93 to 192.

[0268] Embodiment 195. An editing system, A nucleic acid according to any one of embodiments 78 to 92, An editing system comprising a nucleic acid encoding a guide as described in any one of embodiments 93 to 177 or 190.

[0269] Embodiment 196. The editing system according to Embodiment 194 or 195, wherein the nucleic acid encoding the polypeptide is exogenous mRNA.

[0270] Embodiment 197. The editing system according to Embodiment 194 or 195, wherein the nucleic acid encoding the polypeptide is DNA.

[0271] Embodiment 198. The editing system according to any one of Embodiments 195 to 197, wherein the nucleic acid encoding the guide is DNA.

[0272] Embodiment 199. A composition comprising the editing system described in any one of Embodiments 193 to 198 and an LNP.

[0273] Embodiment 200. A viral vector comprising the editing system described in Embodiment 195.

[0274] Embodiment 201. A method for editing a target nucleic acid, comprising administering the composition described in Embodiment 199 or the viral vector described in Embodiment 200 to a target.

[0275] Embodiment 202. A method for editing a target nucleic acid, comprising contacting a cell with the composition described in Embodiment 199 or the viral vector described in Embodiment 200.

[0276] Embodiment 203. A method for forming double-strand breaks or nicks in a target nucleic acid, comprising contacting a cell with the composition described in Embodiment 199 or the viral vector described in Embodiment 200.

[0277] Embodiment 204. The composition described in Embodiment 199 or the viral vector described in Embodiment 200 for use in therapy.

[0278] The embodiments provided herein relate to guide nucleic acid binders, editing systems comprising guide nucleic acid binders, and methods of using them. In certain embodiments, the guide nucleic acid binder comprises a Cas protein. In certain embodiments, the guide nucleic acid binder comprises a Cas enzyme. In certain embodiments, the guide nucleic acid binder comprises a Cas protein comprising a RuvC domain and an HNH domain. In certain embodiments, the guide nucleic acid binder comprises a class 2 type II Cas protein. In certain embodiments, the guide nucleic acid binder comprises a class 2 type II Cas endonuclease. In certain embodiments, the guide nucleic acid binder comprises a Cas protein comprising a RuvC domain and an HNH domain. In certain embodiments, the guide nucleic acid binder comprises a modified Cas protein ("Cas nickase") having an inactivating mutation in one of its nuclease active sites. In certain embodiments, the guide nucleic acid binder comprises a modified Cas protein ("dead Cas" or "dCas") having inactivating mutations in both nuclease active sites. In certain embodiments, the protein is isolated. In certain embodiments, the protein is manipulated.

[0279] In certain embodiments, the editing system includes a guide nucleic acid binder and a guide. In certain embodiments, the guide consists of a single oligonucleotide and is a single guide, comprising a target recognition region and a protein recognition region. In certain embodiments, the guide is a dual guide, where the first oligonucleotide comprises the first portion of the target recognition region and the protein recognition region, and the second oligonucleotide comprises the second portion of the protein recognition region. In certain such embodiments, the first oligonucleotide is "crRNA" and the second oligonucleotide is "tracrRNA". The target recognition region is a region of oligonucleotide (single guide or crRNA) having a nucleic acid base sequence complementary to an isolength portion of the target sequence in the complementary strand of the target DNA.

[0280] I. Certain nucleic acid binding agents A. A specific polypeptide In certain embodiments, the guide nucleic acid binder is an isolated or manipulated polypeptide. In certain embodiments, the polypeptide consists of a Cas protein or is a fusion protein containing a Cas protein. In certain embodiments, the Cas protein is a Cas enzyme. In alternative embodiments, the Cas protein is a dead Cas protein. In certain embodiments, the polypeptide constituting the Cas protein has one or more domains. In certain embodiments, the domain of the polypeptide is a RuvC domain, which consists of one or more subdomains (e.g., the RuvCI, RuvCII, and / or RuvCIII subdomains described herein). In certain embodiments, the domain of the polypeptide is, for example, an HNH domain described herein. In certain embodiments, the domain of the polypeptide is, for example, a PAM interaction (PI) domain described herein. In certain embodiments, the polypeptide comprises a RuvC domain and an HNH domain. In certain embodiments, the polypeptide comprises a RuvC domain, an HNH domain, and a PI domain. In certain embodiments, the polypeptide comprises a RuvC domain, an HNH domain, a PI domain, and a nuclear localization sequence (NLS). In certain embodiments, the RuvC domain is divided into two or three discontinuous subdomains called RuvCI, RuvCII, and RuvCIII. In certain embodiments, the HNH domain is part of the NUC lobe. In certain embodiments, the polypeptide further includes an REC lobe. In certain embodiments, the REC lobe consists of subdomains corresponding to three REC domains (RECI, RECII, and RECIII). In certain embodiments, RECI is divided into two discontinuous subdomains (RECIa and RECIb). ​​In certain embodiments, the polypeptide includes a bridge helix domain. Representative sequences corresponding to these domains are listed in the table below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0281] In certain embodiments, the polypeptide comprises a region of at least 20, at least 25, or 30 amino acids having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 85. In certain embodiments, the polypeptide comprises a region of at least 20, at least 25, or 30 amino acids having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 85.

[0282] In a particular embodiment, the polypeptide comprises a region of at least 30, at least 40, at least 50, or 55 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 92. In a particular embodiment, the polypeptide comprises a region of at least 30, at least 40, at least 50, or 55 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 92.

[0283] In a particular embodiment, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 94. In a particular embodiment, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 94.

[0284] In a particular embodiment, the polypeptide comprises at least 15, at least 16, at least 17, or 18 amino acid regions having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 97. In a particular embodiment, the polypeptide comprises at least 15, at least 16, at least 17, or 18 amino acid regions having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 97.

[0285] In certain embodiments, the polypeptide comprises a region of at least 30, at least 40, at least 45, or 50 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 104. In certain embodiments, the polypeptide comprises a region of at least 30, at least 40, at least 45, or 50 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 104.

[0286] In a particular embodiment, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 106. In a particular embodiment, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 106.

[0287] In certain embodiments, the polypeptide comprises a region of at least 35, at least 40, at least 45, or 48 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 109. In certain embodiments, the polypeptide comprises a region of at least 35, at least 40, at least 45, or 48 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 109.

[0288] In a particular embodiment, the polypeptide comprises a region of at least 35, at least 40, at least 45, or 47 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 116. In a particular embodiment, the polypeptide comprises a region of at least 35, at least 40, at least 45, or 47 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 116.

[0289] In a particular embodiment, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 118. In a particular embodiment, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 118.

[0290] In certain embodiments, the polypeptide includes a RuvCI domain having at least 70% sequence identity with SEQ ID NO: 85, SEQ ID NO: 97, or SEQ ID NO: 109. In certain embodiments, the polypeptide includes a RuvCI domain having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 85, SEQ ID NO: 97, or SEQ ID NO: 109.

[0291] In certain embodiments, the polypeptide comprises a RuvCII domain having at least 70% sequence identity with SEQ ID NO: 92, SEQ ID NO: 104, or SEQ ID NO: 116. In certain embodiments, the polypeptide comprises a RuvCII domain having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 92, SEQ ID NO: 104, or SEQ ID NO: 116.

[0292] In certain embodiments, the polypeptide comprises a RuvCIII domain having at least 70% sequence identity with SEQ ID NO: 94, SEQ ID NO: 106, or SEQ ID NO: 118. In certain embodiments, the polypeptide comprises a RuvCIII domain having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 94, SEQ ID NO: 106, or SEQ ID NO: 118.

[0293] In certain embodiments, the polypeptide includes a RuvC domain comprising subdomains having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 85, 92, and 94, or SEQ ID NOs. 97, 104, and 106, or SEQ ID NOs. 109, 116, and 118.

[0294] In a particular embodiment, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 93. In a particular embodiment, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 93.

[0295] In a particular embodiment, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 105. In a particular embodiment, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 105.

[0296] In a particular embodiment, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 117. In a particular embodiment, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 117.

[0297] In certain embodiments, the polypeptide includes an HNH domain having at least 70% sequence identity with any of SEQ ID NOs: 93, 105, or 117. In certain embodiments, the polypeptide includes an HNH domain having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NOs: 93, 105, or 117.

[0298] In certain embodiments, the polypeptide comprises a region of at least 70, at least 75, or at least 80 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 87. In certain embodiments, the polypeptide comprises a region of at least 70, at least 75, or at least 80 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 87.

[0299] In a particular embodiment, the polypeptide comprises a region of at least 70, at least 75, or at least 80 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 99. In a particular embodiment, the polypeptide comprises a region of at least 70, at least 75, or at least 80 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 99.

[0300] In a particular embodiment, the polypeptide comprises a region of at least 70, at least 75, or 77 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 111. In a particular embodiment, the polypeptide comprises a region of at least 70, at least 75, or 77 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 111.

[0301] In a particular embodiment, the polypeptide comprises a region of at least 150, at least 175, or at least 200 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 89. In a particular embodiment, the polypeptide comprises a region of at least 150, at least 175, or at least 200 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 89.

[0302] In a particular embodiment, the polypeptide comprises a region of at least 150, at least 175, or at least 200 amino acids having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 101. In a particular embodiment, the polypeptide comprises a region of at least 150, at least 175, or at least 200 amino acids having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 101.

[0303] In a particular embodiment, the polypeptide comprises a region of at least 150, at least 175, or at least 200 amino acids having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 113. In a particular embodiment, the polypeptide comprises a region of at least 150, at least 175, or at least 200 amino acids having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 113.

[0304] In certain embodiments, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 88. In certain embodiments, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 88.

[0305] In a particular embodiment, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 100. In a particular embodiment, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 100.

[0306] In a particular embodiment, the polypeptide comprises a region of at least 50, at least 55, or 60 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 112. In a particular embodiment, the polypeptide comprises a region of at least 50, at least 55, or 60 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 112.

[0307] In a particular embodiment, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 90. In a particular embodiment, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 90.

[0308] In a particular embodiment, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 102. In a particular embodiment, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 102.

[0309] In a particular embodiment, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 114. In a particular embodiment, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 114.

[0310] In certain embodiments, the polypeptide includes a RECIa domain having at least 70% sequence identity with SEQ ID NO: 87, SEQ ID NO: 99, or SEQ ID NO: 111. In certain embodiments, the polypeptide includes a RECIa domain having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 87, SEQ ID NO: 99, or SEQ ID NO: 111.

[0311] In certain embodiments, the polypeptide includes a RECIb domain having at least 70% sequence identity with SEQ ID NO: 89, SEQ ID NO: 101, or SEQ ID NO: 113. In certain embodiments, the polypeptide includes a RECIb domain having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 89, SEQ ID NO: 101, or SEQ ID NO: 113.

[0312] In certain embodiments, the polypeptide includes a RECII domain having at least 70% sequence identity with SEQ ID NO: 88, SEQ ID NO: 100, or SEQ ID NO: 112. In certain embodiments, the polypeptide includes a RECII domain having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 88, SEQ ID NO: 100, or SEQ ID NO: 112.

[0313] In certain embodiments, the polypeptide comprises a RECIII domain having at least 70% sequence identity with SEQ ID NO: 90, SEQ ID NO: 102, or SEQ ID NO: 114. In certain embodiments, the polypeptide comprises a RECIII domain having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 90, SEQ ID NO: 102, or SEQ ID NO: 114.

[0314] In a particular embodiment, the polypeptide comprises a region of at least 500, at least 550, at least 600, or 648 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 91. In a particular embodiment, the polypeptide comprises a region of at least 500, at least 550, at least 600, or 648 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 91.

[0315] In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or 650 amino acids having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 103. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or 650 amino acids having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 103.

[0316] In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, at least 650, or at least 656 amino acids having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 115. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, at least 650, at least 650, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the isolength portion of SEQ ID NO: 115.

[0317] In certain embodiments, the polypeptide comprises an REC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 91, 103, or 115.

[0318] In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or 616 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 96. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or 616 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 96.

[0319] In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or at least 648 amino acids having a sequence that has at least 70% sequence identity with the isolength portion of SEQ ID NO: 108. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, at least 648 amino acids having a sequence that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 108.

[0320] In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or at least 650 amino acids having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 120. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the isolength portion of SEQ ID NO: 120.

[0321] In certain embodiments, the polypeptide comprises an NUC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs. 96, 108, or 120.

[0322] In a particular embodiment, the polypeptide comprises a region of at least 175, at least 200, at least 225, or 244 amino acids having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 95. In a particular embodiment, the polypeptide comprises at least 175, at least 200, at least 225, or 244 amino acids having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 95.

[0323] In a particular embodiment, the polypeptide comprises a region of at least 175, at least 200, at least 225, or 242 amino acids having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 107. In a particular embodiment, the polypeptide comprises at least 175, at least 200, at least 225, or 242 amino acids having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 107.

[0324] In a particular embodiment, the polypeptide comprises a region of at least 175, at least 200, at least 225, at least 250, or 278 amino acids having a sequence having at least 70% sequence identity with the isolength portion of SEQ ID NO: 119. In a particular embodiment, the polypeptide comprises at least 175, at least 200, at least 225, at least 250, or 278 amino acids having a sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the isolength portion of SEQ ID NO: 119.

[0325] In certain embodiments, the polypeptide includes a PI domain having at least 70% sequence identity with any of SEQ ID NOs: 95, 107, or 119. In certain embodiments, the polypeptide includes a PI domain having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NOs: 95, 107, or 119.

[0326] In a particular embodiment, the polypeptide comprises an REC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 91, and an NUC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 96.

[0327] In a particular embodiment, the polypeptide comprises an REC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 103, and an NUC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 108.

[0328] In a particular embodiment, the polypeptide comprises an REC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 115, and an NUC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 120.

[0329] In a particular embodiment, the polypeptide comprises an REC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 91; an NUC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 96; and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 95.

[0330] In a particular embodiment, the polypeptide comprises an REC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 103; an NUC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 108; and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 107.

[0331] In a particular embodiment, the polypeptide comprises an REC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 115; an NUC lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 120; and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 119.

[0332] In certain embodiments, the polypeptide comprises RuvCI-bridge helix-RECIa-RECII-RECIb-RECIII-RuvCII-HNH-RuvCIII-PI, where each (sub)domain has at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the corresponding sequence in Table 1. In certain embodiments, the polypeptide comprises REC lobe-NUC lobe, where each (sub)domain has at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the corresponding sequence in Table 1. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0333] In a particular embodiment, the polypeptide includes a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3186. In a particular embodiment, the polypeptide includes a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3186, and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3187. In a particular embodiment, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3186, and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity with SEQ ID NO: 3187.

[0334] In certain embodiments, the polypeptide includes a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3188. In certain embodiments, the polypeptide includes a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3188, and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3189. In a particular embodiment, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3188, and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity with SEQ ID NO: 3189.

[0335] In a particular embodiment, the polypeptide includes a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3190. In a particular embodiment, the polypeptide includes a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3190, and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3191. In certain embodiments, the polypeptide includes a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3190, and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity with SEQ ID NO: 3191. In certain embodiments, the polypeptide includes a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3192. In a particular embodiment, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3192, and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3193.In certain embodiments, the polypeptide includes a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3192, and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity with SEQ ID NO: 3193. In certain embodiments, the polypeptide includes a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3194. In a particular embodiment, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3194, and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3195. In a particular embodiment, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3194, and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity with SEQ ID NO: 3195. In certain embodiments, the polypeptide includes a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3196.In a particular embodiment, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3196, and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3197. In a particular embodiment, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3196, and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity with SEQ ID NO: 3197.

[0336] In a particular embodiment, the polypeptide includes a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3198. In a particular embodiment, the polypeptide includes a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3198, and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3199. In a particular embodiment, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3198, and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity with SEQ ID NO: 3199.

[0337] In certain embodiments, the polypeptide is a Cas protein. In certain embodiments, the polypeptide is a Cas enzyme. In certain embodiments, the polypeptide is a dead Cas protein. In certain embodiments, the polypeptide is a Cas fusion protein.

[0338] B. Protein Engineering Structure-induced protein engineering and protein engineering methods via directed evolution have been applied to a variety of Cas proteins (see Liu, et al., Trends in Biotechnology, 39(3):262-273, 2021).

[0339] 1. A specific heterogeneous domain In certain embodiments, heterologous domains(s) derived from non-Cas proteins can be added to a Cas protein to add function (see, for example, WO2014 / 152432, WO2016 / 063264, WO2022 / 162247, WO2022 / 140577, US2019 / 0233805) to result in a Cas fusion protein. In certain embodiments, the Cas portion of the fusion protein is used to target the heterologous domain(s) to a desired DNA target. In certain embodiments, the Cas portion of the fusion protein is a dead Cas protein.

[0340] In certain such embodiments, the heterogeneous domain(s) are transcription activators or repressors, or their domains. For example, in some cases, the heterogeneous domain(s) repress transcription (e.g., the heterogeneous domain(s) are transcription repressors, polypeptides that function through the recruitment of transcription inhibitor proteins, polypeptides that result in modifications to target DNA such as methylation, polypeptides that result in the recruitment of DNA modifiers, polypeptides that regulate histones related to target DNA, or polypeptides that recruit histone modifiers such as those that modify histone acetylation and / or methylation). In some cases, the heterogeneous domain(s) increase transcription (e.g., the exogenous domain(s) are transcription activators, polypeptides that act through the recruitment of transcription activator proteins, polypeptides that result in modifications to target DNA such as demethylation, polypeptides that result in the recruitment of DNA modifiers, polypeptides that regulate histones related to target DNA, or polypeptides that recruit histone modifiers such as those that modify histone acetylation and / or methylation).

[0341] In certain embodiments, the Cas fusion protein includes heterologous domains having enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimerization activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity).

[0342] In certain embodiments, heterologous domains (or more) modify a second polypeptide (e.g., a histone) related to the target nucleic acid (e.g., a polypeptide having methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity).

[0343] Examples of polypeptides that can be used to increase transcription include transcription activators such as VP16, VP64, VP48, VP160, MyoD1, HSF1, RTA, SET7 / 9, or their domains, the p65 subdomain (e.g., derived from NFκB), as well as the activation domains of EDLL and / or TAL (e.g., for activity in plants), histone lysine methyltransferases such as SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1, or their domains, JHDM2a / b, UTX, JMJD3 Histone lysine demethylases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK, and other histone acetyltransferases or their domains, as well as DNA demethylases such as TenElevenTranslocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, or their domains, are examples of, but are not limited to, these.

[0344] Examples of polypeptides that can be used to reduce transcription include transcription repressor polypeptides such as Krueppel-associated box (KRAB or SKD) domains, KOX1 repressor domains, and MAD. mSIN3 interaction domain (SID), ERF repressor domain (ERD), SRDX repressor domain (e.g., for repression in plants), histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, RIZ1, or their domains, histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASCI, JMJD2D, JARID1A / RBP2, JARID1B / PLU-I, JARID1C / SMCX, JARID1D / SMCY, histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRTI, SIRT2, HDAC11, or their domains, HhaI DNA Examples include, but are not limited to, DNA methylases such as m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plant), ZMET2, CMTI, CMT2 (plant), or their domains, as well as peripheral mobilization elements such as lamin A and lamin B, or their domains.

[0345] In certain embodiments, heterologous domains possess enzymatic activity that modifies target nucleic acids. Examples of enzymatic activity that can be provided by heterologous domains include nuclease activity, such as that provided by restriction proteins (e.g., Fokl nuclease); methyltransferase activity, such as that provided by methyltransferases (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plant), ZMET2, CMT1, CMT2 (plant)); demethylase activity, such as that provided by demethylases (e.g., 111 translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROSI, etc.); DNA repair activity; DNA damage activity; and deaminase activity (e.g., rat APOBEC1, etc.). Examples of such activities include, but are not limited to, deamination activity provided by cytosine deaminase enzymes, dismutase integrase activity provided by integrases and / or resolvers (e.g., Gin invertase, Gin invertase such as a highly active variant of GinH106Y, human immunodeficiency virus type 1 integrase (IN), Tn3 resolver), transposase activity, recombinase activity provided by recombinases (e.g., the catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0346] In certain embodiments, the heterologous domain has enzymatic activity that modifies proteins related to target nucleic acids (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., histones, RNA-binding proteins, DNA-binding proteins).Examples of enzymatic activity that can be provided by heterologous domains (modifying proteins related to target nucleic acids) include histone methyltransferase (HMT) (e.g., suppressor of plaque expression 3-9 homolog 1 (SUV39Hl, also known as KMT1A)), euchromatin histone lysine methyltransferase 2 (also known as G9A, KMT1C and EHMT2), SUV39H2, ESET / SETDBI, SET1A, SET1B, MLL1-5, ASH1, and SY Methyltransferase activity such as that provided by MD2, NSD1, DOT1L, Pr-SET7 / 8, SUV4-20H1, EZH2, RIZ1), histone demethylase (e.g., lysine demethylase 1A (also known as KDM1A, LSD1), JHDM2a / b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, Demethylase activity such as that provided by UTX, JMJD3), acetyltransferase activity such as that provided by histone acetylase transferases (e.g., catalytic core / fragment of human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, HBO1 / MYST2, HMOF / MYST1, SRC1, ACTR, P160, CLOCK), histone deacetylase (e.g., Examples of deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity, as provided by HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRTI, SIRT2, HDACII, etc., include, but are not limited to, those provided by HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRTI, SIRT2, HDACII.

[0347] Domains that have been added to Cas proteins include FokI for highly specific genome editing (see, e.g., WO2014 / 152432), transcriptional repressor domains for gene expression regulation (e.g., KRAB; see, e.g., Alerasool, et al., Nature Methods, 1093-1096, 2020) and transcriptional activator domains (e.g., VP64-e.g., WO2014 / 197748, p65, and RTA), histone modifying enzymes such as methyltransferases for epigenetic editing (see, e.g., WO2016 / 103233), and fluorescent proteins for genome imaging (see, e.g., Trends in Biotechnology, 39(3):262-273, 2021). One or more of these polypeptides can be added to the Cas protein at either the N-terminus or C-terminus. In certain embodiments, multiple domains are attached to the Cas protein at the C-terminus, N-terminus, or both (see, for example, WO2019 / 204766). Additional repressor domains that can be attached to the Cas protein are provided, for example, in WO2022 / 140577, Alerasool, et al., Nature Methods, 1093-1096, 2020.

[0348] For some examples of the above polypeptides used in the context of fusion with Cas9, zinc fingers, and / or TALE proteins (for site-directed targeted nucleic acid modifications, transcriptional regulation, and / or targeted protein modifications, e.g., histone modifications), see, for example, Nomura et al., J Am Chem Soc. 2007 Jul 18;129(28):8676-7, Rivenbark et al., Epigenetics. 2012 Apr;7(4):350-60, Nucleic Acids Res. 2016 Jul 8;44(12):5615-28, Gilbert et al., Cell. 2013 Jul 18;154(2):442-51, Kearns et al., Nat Methods. 2015 May;12(5):401-3, Mendenhall et al., Nat Biotechnol. 2013. Dec;31(12):1133-6, Hilton et.al., Nat Biotechnol.2015 May;33(5):510-7, Gordley et.al., Proc Natl Acad Sci US A.2009 Mar 31;106(13):5053-8, Akopian et.al., Proc Natl Acad Sci US A.2003 Jul 22;100(15):8688-91, Tan et.,al.,J Virol.2006 Feb;80(4):1939-48, Tan et.al.,Proc Natl Acad Sci US A.2003 Oct 14;100(21):11997-2002, Papworth et.al.,Proc Natl Acad Sci US A.2003 Feb 18;100(4):1621-6, Sanjana et.al., Nat Protoc.2012 Jan 5;7(1):171-92, Beerli et.al., Proc Natl Acad Sci USA.1998 Dec 8;95(25):14628-33, Snowden et.al., Curr Biol.2002 Dec 23;12(24):2159-66, Xu et.al.,Xu et.al.,Cell Discov.2016 May 3;2:16009;Komor et al.,Nature.2016 Apr 20;533(7603):420-4;Chaikind et.al.,Nucleic Acids Res.2016 Aug 11;Choudhury et.al.,Oncotarget.2016 Jun 23.Du et al.,Cold Spring Harb Protoc.2016 Jan 4;Pham et al.,Methods Mol Biol.2016;1358:43-57;Balboa et al.,Stem Cell Reports.2015 Sep 8;5(3):448-59;Hara et al.,Sci Rep.2015 Jun 9;5:11221;Piatek et al.,Plant Biotechnol J.2015 May;13(4):578-89;Hu et al.,Nucleic Acids Res.2014 Apr;42(7):4375-90;Cheng et al.,Cell Res.2013 Oct;23(10):1163-71 Cheng et al., Cell Res Oct;l0(l0):977-9.

[0349] 2. This snowflake snowflake In certain embodiments, Cas proteins may contain one or more nuclear localization sequences. Nuclear localization sequences are short sequences of amino acids that facilitate protein uptake into the cell nucleus. A variety of eukaryotic nuclear localization signal (NLS) sequences have been used to improve protein nuclear uptake (see, e.g., Lu, et al., Cell Comm. and Signalling, 2021). NLSs may be located at the N-terminus and / or C-terminus of a polypeptide, or at any point within the polypeptide sequence. The classic monopartite NLS has the sequence K(K / R)X(K / R) (where K is lysine and X can be any amino acid). The bipartite NLS contains two clusters of 2-3 positively charged amino acids separated by a region of 9-12 amino acids containing several prolines, and R / K(X) 10-12 It has a consensus sequence of KRXK (where R is arginine, K is lysine, and X is any amino acid). In certain embodiments, the Cas protein may comprise one or more nuclear localization sequences or variants thereof selected from the following table. [Table 3]

[0350] 3. Modified Cas variants with PAM specificity The Cas protein binds to target DNA at a sequence defined by a complementation region between the target recognition region of the guide and the target DNA. Site-specific binding (and / or cleavage) of the Cas protein to the double-stranded target DNA typically occurs at a site determined by both (i) hybridization of the target recognition region of the guide to the target DNA and (ii) the presence of a short motif (referred to as a PAM or protospacer-adjacent motif) in the non-complementary strand of the DNA. In certain embodiments, the PAM of the Cas protein is located immediately 5' to the protospacer sequence. In certain embodiments, the PAM of the Cas protein is located immediately 3' to the protospacer sequence.

[0351] In particular, methods for manipulating the PAM interaction domain (PI domain) of Cas proteins, including SpCas9, SaCas9, StlCas9, FnCas9, and AsCas12a, have been previously described (see, for example, Table 1 in WO2016 / 141224, Liu, et al., Trends in Biotechnology, 39(3):262-273, 2021).

[0352] A bacterial selection system can be used to optimize Cas protein variants that have a non-native PI domain configured to recognize variant PAM. Bacteria are transfected with a plasmid encoding an inducible toxic gene, as well as a plasmid encoding a Cas protein variant. After induction of the toxic gene, the only surviving bacteria are those in which the Cas protein variant cleaves the toxic gene. Using this method, a large library of Cas proteins with randomized mutations in the PI domain can be screened against a large randomized PAM library. For the surviving clones, the PAM specificity of the evolved Cas protein can then be profiled using a bacterial-based site depletion assay. These methods have been previously used to identify PI domains with novel PAM-recognizing sequences in SpCas9, SaCas9, StlCas9 (Kleinstiver, Nature, 523(7561):481-485, 2015), and SaCas9 (Kleinstiver, Nature Biotech., 33(12):1293-1298, 2015). Similarly, selection systems using phage-assisted continuous evolution (PACE) have been used to identify Cas9 variants with extended PAM recognition (Hu, et al., Nature, 556:57-63, 2018; Miller, et al., Nature Biotech., 38:471-481, 2020).

[0353] Another method previously used to design alternative PAM interaction domains that recognize variant PAM is structure-inducible protein engineering. This method identifies amino acid residues that form contact with target DNA via crystal structure, and then Cas protein variants containing mutations in these amino acids are tested for activity. Using these methods, variants of saCas9 with reduced off-target editing have been identified (Tan, et al., PNAS, 116(42):20969-20976, 2019), and similarly, variants of AsCas12a have been identified (Kleinstiver, et al., Nature Biotech., 37(3):276-282, 2019). This method has also been used to identify variants of FnCas9 with broader PAM recognition sequences (Hirano, et al., Cell, 164:950-961).

[0354] A further method used to generate Cas protein variants that recognize variant PAM is to create domain-swap chimeras by directly substituting the PI domain of a Cas protein with the PI domain of a closely related orthologous Cas protein. In earlier studies, swapping the PI domain of SpCas9 with the PI domain of St3Cas9 was shown to generate a variant of St3Cas9 that cleaves target DNA using canonical SpCas9 5'-NGG-3'PAM (Nishimasu, et al, Cell, 156(5):935-949, 2014). In a similar manner, several Cas9 variants that recognize variant PAM were identified by swapping the PI domain with a closely related Cas protein (Ma, et al., Nature Comm., 10:560, 2019).

[0355] A specific nucleic acid that codes for C. polypeptide. 1. A specific nucleic acid In certain embodiments, nucleic acids encoding polypeptides are provided herein. In certain embodiments, the polypeptide is or is part of a guide nucleic acid binder. In certain embodiments, the polypeptide comprises or consists of a Cas protein. In certain embodiments, the polypeptide comprises or consists of an ION-Cas protein.

[0356] In certain embodiments, the nucleic acid is DNA. In certain embodiments, the nucleic acid is RNA. In certain embodiments, the nucleic acid is exogenous mRNA.

[0357] In certain embodiments, the exogenous mRNA includes a coding region (e.g., an open reading frame (ORF)) encoding a polypeptide sequence, a cap, and one or more non-coding regions. In certain embodiments, the exogenous mRNA includes a cap, a 5'UTR, a 3'UTR, a coding region, a poly(A) tail, and optionally one or more introns. The mRNA may contain nucleotides selected from adenosine, guanosine, cytosine, uridine, and N1-methylpseudridine, and optionally, nucleotides selected from adenosine, guanosine, cytosine, and uridine.

[0358] Naturally occurring eukaryotic mRNA molecules may contain non-coding regions, including but not limited to untranslated regions (UTRs), a 5' cap structure, and a 3' poly(A) tail, at their 5' end (5'UTR) and / or their 3' end (3'UTR). Exogenous mRNA may be configured to contain such regions, which facilitates cellular processing and translation. In certain embodiments, a formulation (e.g., LNP) may be configured to deliver exogenous mRNA having an open reading frame encoding a polypeptide. In certain embodiments, a method is provided for translating exogenous mRNA in a cell.

[0359] Exogenous mRNA may contain repeat regions of consecutive nucleosides, such as adenosine nucleosides (e.g., poly(A) tails). A poly(A) tail is a region at the 3' end of the 3' UTR containing multiple consecutive adenosine monophosphates. Poly(A) tails are thought to protect mRNA from enzymatic degradation (e.g., in the cytoplasm), assist in transcription termination and / or mRNA export from the nucleus, and translation. A poly(A) tail may contain 10 to 300 consecutive adenosine nucleosides. For example, exogenous mRNA may have repeat regions of 50 to 100, 50 to 150, 50 to 200, 100 to 250, 120 to 160, or 200 to 300 adenosine monophosphates at its 3' end.

[0360] 2. Capping base Eukaryotic mRNA, as well as RNA viral genomes, contain a 7-methylguanosine (m7G) cap at the 5' end of the mRNA sequence, which, during in vitro mRNA transcription, binds to the outermost 5' mRNA nucleotide via a 5',5' triphosphate crosslink (ppp). The cap structure plays an essential role in mRNA translation by recruiting translation initiation factors and can incorporate different 5' caps into naturally occurring mRNA. Cap0 protects endogenous mRNA from nuclease attack and is also involved in nuclear export and translation initiation. Both Cap1 and Cap2 are two 5' caps containing an additional methyl group on a second or third ribonucleotide. Additional modifications of Cap1 and Cap2 are thought to reduce immunogenicity compared to Cap0.

[0361] Exogenous mRNA may contain specific capping groups, such as those described herein or those known in the art. Examples of 5' capping groups include "Cap0": m7G(5')ppp(5')N, "Cap1": m7G(5')ppp(5')(2'OMeN), and "Cap2": m7G(5')ppp(5')(2'OMeN)(2'OMeN), where m7G represents a guanosine nucleoside methylated at position 7 and having a free 3'-OH group, (5') represents a 5' linkage, p is a phosphate linkage, each N is independently a nucleoside (e.g., guanosine or adenosine), and (2'OMeN) is independently a 2'-O-methyl nucleoside (e.g., 2'-O-methylguanosine or 2'-O-methyladenosine). The first nucleoside adenosine after the cap can also be methylated at its N6 position.

[0362] Specific capping groups include (m7(3′OMeG)(5′)ppp(5′)(2′OMeA)pG, 3'-O-Me-m7G(5')ppp(5')G ("ARCA" cap), G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, and m7G(5')ppp(5')(2'OMeN)pG ("CleanCap®"), where m7(3'OMeG) is methylated at position 7, indicating a guanosine nucleoside with a 3'-O-methyl group. Commercial sources are available for some of these groups (e.g., New England BioLabs, Ipswich, MA and TriLink Biotechnologies, San Diego, CA). 5' capping of exogenous mRNA is performed, for example, by Vaccinia. Capping can be completed post-transcriptionally using Vims capping enzymes. The Cap1 structure can be generated using both Vaccinia Vims capping enzymes and 2'-O methyltransferase. The Cap2 structure can be generated from the Cap1 structure, and then the third nucleotide from the 5' end is 2'-O methylated using 2'-O methyltransferase. The Cap3 structure can be generated from the Cap2 structure, and then the fourth nucleotide from the 5' end is 2'-O methylated using 2'-O methyltransferase. The enzymes may be derived, for example, from a recombinant source.

[0363] 3. Poly(A) Tail The 3'-poly(A) tail is a region of consecutive adenine nucleotides at the 3' end of transcribed mRNA. In certain embodiments, the 3'-poly(A) tail contains 1 to 400 adenine nucleotides. In certain embodiments, the 3'-poly(A) tail contains unmodified adenine nucleosides linked by phosphodiester nucleoside bonds.

[0364] In some embodiments, the exogenous mRNA contains a stabilizing element. This stabilizing element may be a histone stem-loop. A stem-loop-binding protein (SLBP), a 32 kDa protein, has been identified. It is thought to associate with the histone stem-loop at the 3' end of histone messages in both the nucleus and cytoplasm, facilitating efficient 3' end processing and translation of histone pre-mRNA. In some embodiments, the histone stem-loop sequence contains 15–45 nucleotides in length.

[0365] In some embodiments, the exogenous mRNA comprises a coding region, at least one histone stem-loop, and optionally a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal is thought to increase the expression of the encoded protein. In some embodiments, the encoded protein is not a histone protein, a reporter protein (e.g., luciferase, GFP, EGFP, β-galactosidase, EGFP), or a marker or selection protein (e.g., alpha-globin, galactokinase, and xanthine:guanine phosphoribosyltransferase (GPT)).

[0366] 4. Optimization of code areas and arrays The coding region contains a contiguous sequence beginning with a start codon (e.g., methionine (AUG)) and ending with a stop codon (e.g., UAA, UAG, or UGA), and may contain one or more nucleotide sequences that code for an amino acid. Generally, the coding region codes for a polypeptide, which, when administered to a subject, forms a protein in vivo.

[0367] In certain embodiments, the coding region may include selected nucleotide codons and a codon-optimized ORF to improve one or more properties of exogenous mRNA or coding protein. For example, the coding region of an exogenous mRNA sequence may be codon-optimized. In some embodiments, codon optimization may be used to match the codon frequencies of the target organism and the host organism to ensure proper folding, to increase the stability of the mRNA, or to reduce secondary structures by biasing the GC content, minimizing tandem repeat codons or base runs that may impair gene construction or expression, customizing transcription and translation regulatory regions, inserting or removing protein transport sequences, adding or removing post-translational modification sites of the encoded protein (e.g., glycosylation sites), adding, removing, or shuffling protein domains, inserting or deleting restriction sites, modifying ribosome binding sites and mRNA degradation sites, adjusting the translation rate to ensure proper folding of various domains of the protein, or generally to reduce or eliminate problematic secondary structures within a polynucleotide. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or proprietary methods. In certain embodiments, the code region sequence is optimized using optimization algorithms provided herein or optimization algorithms known in the art.

[0368] In certain embodiments, the coding region shares less than 95%, less than 90%, less than 80%, or less than 70% sequence identity with a naturally occurring sequence ORF or wild-type sequence ORF (e.g., a naturally occurring mRNA sequence or wild-type mRNA sequence that codes for a protein), or a range of values ​​between them.

[0369] In some embodiments, exogenous mRNA may have enhanced G / C levels. The G / C content of a nucleic acid molecule (e.g., mRNA) can affect RNA stability. RNA with increased amounts of guanine (G) and / or cytosine (C) residues may be more stable than RNA containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. See, for example, WO02 / 098443.

[0370] The coding region contains at least one start codon (AUG nucleotide triplet) in its most 5' nucleotide and at least one stop codon (UAG, UAA, or UGA nucleotide triplet) in its most 3' nucleotide. In certain embodiments, the exogenous mRNA contains two or more stop codons (e.g., 2 to 10 stop codons).

[0371] Exemplary codons of certain amino acids are known in the art.

[0372] 5. Chemical modifications for exogenous mRNA In some embodiments, the compositions of this disclosure include exogenous mRNA comprising modified nucleotides or nucleosides (other than A, C, G, and U linked solely by phosphodiester bonds; excluding the 5' cap). Such modified nucleotides and nucleosides may be naturally occurring or non-naturally occurring. Such modifications may include those described herein with respect to oligonucleotides.

[0373] In certain embodiments, the naturally occurring modified nucleotides or nucleotides described herein are generally known or recognized in the art. A non-limiting list of such naturally occurring modified nucleotides and nucleotides can be found, among other things, in the widely recognized MODOMICS database. In certain embodiments, the exogenous mRNA includes a modified nucleoside described in one or more of the following US applications: PCT / US2012 / 058519, PCT / US2013 / 075177, PCT / US2014 / 058897, PCT / US2014 / 058891, PCT / US2014 / 070413, PCT / US2015 / 36773, PCT / US2015 / 36759, PCT / US2015 / 36771, or PCT / IB2017 / 051367 (all of which are incorporated herein by reference). Thus, the exogenous mRNA may include an unmodified nucleoside, a modified nucleoside, or a combination thereof. In certain embodiments, each nucleoside in exogenous mRNA is modified in the same way as other nucleosides of the same type, for example, all uridine nucleosides are present as N1-methylpseudolidine nucleosides. The modification may provide reduced degradation and / or reduced immunogenicity compared to RNA containing only unmodified nucleosides.

[0374] In certain embodiments, the exogenous mRNA comprises a modified nucleoside selected from 1-methylpseudridine, 1-ethylpseudridine, 5-methoxyuridine, 5-methylcytidine, and pseudouridine. In certain embodiments, the exogenous mRNA comprises a modified nucleoside selected from 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudridine, 5-methylcytidine, and 5-methoxycytidine. In certain embodiments, the exogenous mRNA comprises a modified nucleoside selected from any combination of two or more (e.g., 2, 3, 4) of the modified nucleic acid bases of this paragraph.

[0375] In certain embodiments, the exogenous mRNA contains 1-methylpseudrulin at one or more (e.g., all) uridine nucleosides. In certain embodiments, the exogenous mRNA contains 1-methylpseudrulin at one or more (e.g., all) uridine positions of the nucleic acid and 5-methylcytidine at one or more (e.g., all) cytidine positions of the nucleic acid.

[0376] In certain embodiments, the exogenous mRNA contains pseudouridine at one or more (e.g., all) uridine nucleosides. In certain embodiments, the exogenous mRNA contains pseudouridine at one or more (e.g., all) uridine positions of the nucleic acid and 5-methylcytidine at one or more (e.g., all) cytidine positions of the nucleic acid.

[0377] In certain embodiments, the exogenous mRNA contains unmodified uridines at one or more (e.g., all) uridine positions of the nucleic acid.

[0378] In a particular embodiment, all nucleotides of a certain type in exogenous mRNA (or in a sequence region thereof) are modified nucleotides, and the modification type may be any one of nucleotides A, G, U, or C, or any combination thereof A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0379] 6. Untranslated Area (UTR) In wild-type mRNA, certain regions of the nucleic acid can be transcribed into RNA but not translated. In the exogenous mRNA described herein, the 5'UTR begins at the transcription start site and may be followed by the start codon at the beginning of the coding region, but does not contain the start codon. The 3'UTR following the stop codon may contain the transcription termination signal. The 5'UTR and 3'UTR do not code for proteins (they are non-coding regions).

[0380] In certain embodiments, the presence of a UTR enhances the stability of exogenous mRNA or induces downregulation of exogenous mRNA in undesirable sites or tissues. A variety of 5'UTR and 3'UTR sequences are known and available in the art.

[0381] The 5'UTR is the region of mRNA directly upstream (5' side) from the start codon (the first codon of the mRNA transcript translated by the ribosome). In certain embodiments, the 5'UTR may provide translation initiation and / or form secondary structures involved in the binding of elongation factors. The 5'UTR may contain an initiation sequence such as a Kozak sequence. The Kozak sequence, also called the Kozak consensus sequence, is thought to be involved in the initiation of ribosome translation. In certain embodiments, the Kozak sequence contains AUGG. In certain embodiments, the Kozak sequence is GCCRCCAUGG (SEQ ID NO: 73) or CRCCAUGG, where R is a purine nucleoside (adenosine or guanosine).

[0382] In certain embodiments of this disclosure, the 5'UTR is an unmodified UTR, i.e., one that is found in nature. In other embodiments, the 5'UTR is a modified UTR, i.e., one that does not exist in nature. In certain embodiments, the modified UTR increases gene expression compared to its unmodified counterpart. Examples of 5'UTRs include Xenopus laevis or human-derived α-globin or β-globin (e.g., U.S. Patent No. 9012219), human cytochrome β-245a polypeptide, and hydroxysteroid (17b) dehydrogenase, as well as tobacco etch virus (e.g., US9012219), CMV earliest 1 (IE1) gene (US2014 / 0206753, WO2013 / 185069), sequence GGGAUCCUACC (SEQ ID NO: 74) (WO2014 / 144196), and 5'UTRs described in U.S. Patent Application Publication No. 2010 / 0293625 or WO2015 / 085318. In another embodiment, the 5'UTR of the TOP gene is the 5'UTR of the TOP gene lacking the 5'TOP motif (oligopyrimidine tract) (e.g., WO / 2015 / 101414, WO2015 / 101415, WO / 2015 / 062738, WO2015 / 024667, WO2015 / 024667), and is derived from the 5'UTR element of the ribosomal protein Large32 (L32) gene. The ment (WO / 2015 / 101414, W02015 / 101415, WO / 2015 / 062738), the 5'UTR element derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (WO2015 / 024667), or the 5'UTR element derived from the 5'UTR of ATP5A1 (WO2015 / 024667) can be used. In some embodiments, the internal ribosome entry site (IRES) is used instead of the 5'UTR.

[0383] Generally, exogenous mRNA may contain UTRs derived from any suitable gene. Naturally occurring UTRs or portions thereof may be oriented in the same orientation as the selected transcript, or their orientation or position may be modified. Thus, 5'UTRs or 3'UTRs may be inverted, shortened, or lengthened. Reference UTRs (e.g., naturally occurring UTRs) may be modified by the inclusion of additional nucleotides, deletion or swapping of nucleotides, or transposition of nucleotides or their sequences.

[0384] In certain embodiments, the 3'UTR sequence may contain adenosine and uridine-containing repeats. Such AU-rich sequences are thought to provide a high turnover rate. Based on their sequence and functional characteristics, AU-rich elements (AREs) can be divided into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUASS nonomers, where S is adenosine or uridine in the sequence. Molecules containing this type of ARE include GM-CSF and TNF-α. Class III AREs are less clearly defined. These U-rich regions do not contain the AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Inclusion of a HuR-specific binding site in the 3'UTR is thought to provide in vivo message stabilization. In a particular embodiment, 3'UTR includes an iterative ARE.

[0385] Unmodified (natural) and modified (unnatural) 3'UTR sequences are known in the art. UTRs known in the art include Xenopus β-globin UTR and human β-globin UTR (9012219, US2011 / 0086907), modified β-globin constructs (US2012 / 0195936, WO2014 / 071963), α2-globin, α1-globin, UTR (WO2015 / 101415, WO2015 / 024667), CYBA (Ferizi et al. ah, 2015), and albumin (Thess et al.). This includes globin UTRs (ah, 2015), bovine or human growth hormone (wild-type or modified) (WO2013 / 185069, US2014 / 0206753, WO2014 / 152774), rabbit β-globin and hepatitis B virus (HBV), α-globin 3'UTR and viral VEEV 3'UTR sequences, sequence UUUGAAUU (WO2014 / 144196), human and mouse ribosomal protein rps9 3'UTR (WO2015 / 101414), FIG4 (WO2015 / 101415), and human albumin 7 (WO2015 / 101415).

[0386] The untranslated region may also include upward-adjusting motifs, such as translation enhancer elements (TEEs). Non-limiting examples of TEEs include WO1999 / 024595, WO2012 / 009644, WO2009 / 075886, WO2007 / 025008, WO1999 / 024595, European Patent Publications EP2610341A1 and EP2610340A1, and U.S. Patents US6310197, US6849405, and US74562. This may include those described in U.S. Patent Publication No. 73, U.S. Patent Publication No. US2009 / 0226470, U.S. Patent Publication No. US2011 / 0124100, U.S. Patent Publication No. US2007 / 0048776, U.S. Patent Publication No. US2009 / 0093049, or U.S. Patent Publication No. US2013 / 0177581 (each of which is incorporated herein by reference in whole).

[0387] In certain embodiments, the 5'UTR or 3'UTR may include one or more additional functional regions selected from upregulatory regions or ribosome-binding regions. In certain embodiments, the functional region is an upregulatory region (e.g., a TEE). In certain embodiments, the TEE is known in the art, for example, in U.S. Patent Application No. 2009 / 0226470. In certain embodiments, the exogenous mRNA includes a number of upregulatory motifs (e.g., two, three, four, five, or more) that may be the same as or different from one another.

[0388] In certain embodiments, the exogenous mRNA includes a ribosome-binding region, such as an internal ribosome entry site (IRES). The IRES may be, for example, those described in U.S. Patent No. 7468275 and International Patent Publication No. WO2001 / 055369 (each of which is incorporated herein by reference in whole). In certain embodiments, the IRES is known in the art (e.g., WO2014 / 081507).

[0389] In certain embodiments, the exogenous mRNA may include a double, triple, or quadruple UTR, such as a 5'UTR or a 3'UTR. As used herein, a “double” UTR means that two copies of the same UTR are included consecutively or substantially consecutively. For example, the exogenous mRNA may include a double betaglobin 3'UTR, as described in U.S. Patent Publication 2010 / 0129877, which is incorporated herein by reference in its entirety.

[0390] In certain embodiments, a 5'UTR or 3'UTR may contain a repeating set of functional sequences, such as AA, ABAB, AABB, or ABCABC, or variants thereof (where each letter A, B, and C represents a different functional domain). The pattern may be repeated one, two, three, or more times.

[0391] In general, any 5'UTR sequence and any 3'UTR sequence can be combined with a specific exogenous mRNA.

[0392] Other non-coding sequences may also be used as regions or subregions within exogenous mRNA. For example, introns or portions of intron sequences may be incorporated into regions of nucleic acids of this disclosure. In certain embodiments, inclusion of intron sequences may increase protein production and nucleic acid levels.

[0393] 7. Length In certain embodiments, exogenous mRNA contains 200 to 3,000 nucleotides. For example, exogenous mRNA may contain 200 to 500, 200 to 1,000, 200 to 1,500, 200 to 3,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,500 to 3,000, or 2,000 to 3,000 nucleotides. Unless otherwise indicated, exogenous mRNA contains consecutive nucleosides.

[0394] 8. Preparation In some embodiments, the RNA transcript is produced using a non-amplified linearized DNA template in an in vitro transcription reaction. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of RNA polynucleotides.

[0395] An in vitro transcription system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase. NTPs may be purchased from a supplier or synthesized as described herein according to methods known in the art.

[0396] In vitro transcription of RNA is known in the art and is described in International Publication No. WO2014 / 152027 (the entire publication is incorporated herein by reference). In certain embodiments, exogenous mRNA is prepared according to one or more of the methods described in WO2018 / 053209 and WO2019 / 036682 (each of which is incorporated herein by reference).

[0397] 9. Purification The purification of exogenous mRNA described herein may include steps including nucleic acid cleanup, quality assurance, and quality control. Cleanup may be performed by methods known in the art, for example, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark), or by HPLC-based purification methods, for example, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term “purified,” when used with respect to exogenous mRNA, means that it has been isolated from at least one contaminant. “Contaminant” means any substance that makes another unsuitable, impure, or degraded substance. Therefore, purified nucleic acids (e.g., DNA and RNA) exist in a different form or configuration than those found in nature, or in a different form or configuration than those that existed before being subjected to the processing or purification method.

[0398] Quality assurance and / or quality control checks may be performed using methods such as gel electrophoresis, UV absorbance, or analytical HPLC, but are not limited to these.

[0399] In some embodiments, nucleic acids may be sequenced by methods including, but not limited to, reverse transcriptase-PCR.

[0400] II. A specific oligonucleotide In certain embodiments, oligonucleotides comprising bound nucleosides are provided herein. In certain embodiments, a single guide comprises an oligonucleotide. In certain embodiments, a dual guide comprises two oligonucleotides. The oligonucleotide may be an unmodified oligonucleotide or a modified oligonucleotide. A modified oligonucleotide comprises at least one modification to the unmodified nucleic acid. That is, a modified oligonucleotide comprises at least one modified nucleoside (including a modified sugar moiety and / or a modified nucleic acid base), and / or at least one modified nucleoside bond.

[0401] A. A specific modified nucleoside Modified nucleosides contain either a modified sugar moiety, a modified nucleic acid base, or both.

[0402] 1. A specific sugar portion In certain embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety. In certain embodiments, the modified sugar moiety is a bicyclic or tricyclic sugar moiety. In certain embodiments, the modified sugar moiety is a sugar surrogate. Such a sugar surrogate may include one or more substitutions corresponding to other types of modified sugar moieties.

[0403] In certain embodiments, the modified sugar moiety is a non-bicyclic modified furanosyl sugar moiety containing one or more substituents, including substituents at the 2', 3', 4', and / or 5' positions (as numbered below), but not limited to these. [ka]

[0404] In certain embodiments, the modified furanosyl sugar moiety is a ribosyl sugar moiety that is not an unmodified sugar moiety (i.e., an unmodified RNA or unmodified DNA moiety). In certain embodiments, the modified furanosyl sugar moiety is a xylosyl, lyxosyl, or arabinosyl sugar moiety.

[0405] In certain embodiments, the non-bicyclic modified sugar moiety is a 2'-substituted sugar moiety containing a substituent at the 2' position. In certain embodiments, one or more non-crosslinked substituents of the non-bicyclic modified sugar moiety are branched. Examples of suitable substituents at the 2' position of the modified sugar moiety include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE" or "O-methoxyethyl"). In certain embodiments, the 2'-substituents are halo, allyl, amino, azide, SH, CN, OCN, CF3, OCF3, C1-C 10 Alkyl, C1-C 10 Substitutive alkoxy, C1-C 10 Alkyl, C1-C 10 Substitutive alkyl, S-alkyl, N(R m )-alkyl, O-alkenyl, S-alkenyl, N(R m )-Alkenyl, O-Alkinyl, S-Alkinyl, N(R m )-Alkynyl, O-Alkyrenyl-O-Alkyl, Alkynyl, Alkalyl, Aralkyl, O-Alkalyl, O-Aralkyl, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ), or OCH2C(=O)-N(R m )(R n ) are selected from, and in the formula, each R m and R n However, independently, H, an amino protecting group, or substituted or unsubstituted C1-C 10These are alkyl, O(CH2)2ON(CH3)2 ("DMAOE"), or 2'-O(CH2)2O(CH2)2N(CH3)2 ("DMAEOE"). Synthetic methods for some of these 2'-substituents can be found, for example, in Cook et al. US6,531,584, Cook et al. US5,859,221, and Cook et al. US6,005,087. Certain embodiments of these 2'-substituents are independently halo, cyano, OR a2 NO2, NH2, NHR a2 , N(R a2 )2, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, C6-C 10 Aryl, heteroaryl, heterocyclyl, C1-C6 alkylene-NH2, C1-C6 alkylene-NHR a2 , C1-C6 alkylene-N(R a2 )2, C(O)R a3 , C(O)OR a3 C(O)NHR a3 C(O)N(C1-C4 alkyl)R a3 , SR a3 S(O)2R a3 S(O)R a3 NHC(O)R a3 N(C1-C4 alkyl)C(O)R a3 NHS(O)R a3 N(C1-C4 alkyl)S(O)R a3 NHS(O)2R a3 , and N(C1-C4 alkyl)S(O)2R a3 It may be further substituted with one or more substituents selected from, each R a2 However, independently, C2-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Cycloalkyl, C6-C 10 Selected from aryl, heteroaryl, and heterocyclyl, each R a3 However, independently, hydrogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, C3-C 10 Cycloalkyl, C6-C 10The molecule is aryl, heteroaryl, or heterocyclyl. In certain embodiments, the sugar moiety contains two of the above substituents at the 2' position. In certain embodiments, the sugar moiety contains a 2'-fluoro substituent and a second 2'- substituent.

[0406] In certain embodiments, the 2'-substituted sugar moiety is F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2 and N-substituted acetamides (OCH2C(=O)-N(R m )(R n The formula includes a non-crosslinked 2'- substituent selected from )), where each R m and R n These are independently H, an amino protecting group, or substituted or unsubstituted C1-C 10 It is alkyl. In certain embodiments, the 2'-substituted sugar moiety includes a non-crosslinked 2'-substituent selected from F, OCF3, OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, O(CH2)2ON(CH3)2 ("DMAOE"), O(CH2)2O(CH2)2N(CH3)2 ("DMAEOE"), and OCH2C(=O)-N(H)CH3 ("NMA").

[0407] In certain embodiments, the 2'-substituted sugar moiety includes a 2'-substituted substituent selected from F, OCH3, and OCH2CH2OCH3.

[0408] In certain embodiments, the modified furanosyl sugar moiety, and the nucleoside incorporating such a modified furanosyl sugar moiety, are further defined by their stereochemical configuration. For example, the 2'-deoxyfuranosyl sugar moiety (i.e., the 2'-(H)H furanosyl sugar moiety) can be in seven isomer configurations other than the naturally occurring β-D-deoxyribosyl configuration. Such modified sugar moieties are described, for example, in WO2020 / 072991 (incorporated herein by reference). The 2'-modified sugar moiety has an additional stereocenter at the 2' position compared to the 2'-deoxyfuranosyl sugar moiety, and therefore such a sugar moiety has a total of 16 possible stereochemical configurations. Unless otherwise specified, the modified furanosyl sugar moieties described herein are in the β-D-ribosyl stereochemical configuration.

[0409] In certain embodiments, the non-bicyclic modified sugar moiety includes a substituent at the 4' position. Examples of suitable substituents at the 4' position of the modified sugar moiety include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al. WO2015 / 106128.

[0410] In certain embodiments, the non-bicyclic modified sugar moiety includes a substituent at the 3' position. Suitable substituents for the 3' position of the modified sugar moiety include, but are not limited to, alkoxy (e.g., methoxy) and alkyl (e.g., methyl, ethyl).

[0411] In certain embodiments, the non-bicyclic modified sugar moiety includes a substituent at the 5' position. Suitable substituents for the 5' position of the modified sugar moiety include, but are not limited to, vinyl, alkoxy (e.g., methoxy), alkynyl, allyl, and alkyl (e.g., methyl (R or S), ethyl (R or S)).

[0412] In certain embodiments, the non-bicyclic modified sugar moiety comprises two or more non-crosslinked sugar substituents, for example, the 2'-F-5'-methyl sugar moiety described in US2010 / 0190837 by Migawa et al. (incorporated herein by reference), or alternative 2'- and 5'-modified sugar moieties described in US2013 / 0203836 by Rajeev et al.

[0413] Certain modified sugar moieties are bicyclic sugar moieties containing substituents that bridge two atoms of a furanosyl ring to form a second ring. In certain embodiments, the bicyclic sugar moiety includes a bridge between a 4'-position furanose ring atom and a 2'-position furanose ring atom. Examples of such 4'-to-2' bridging sugar substituents include 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' ("LNA"), 4'-CH2-S-2', 4'-(CH2)2-O-2' ("ENA"), 4'-CH(CH3)-O-2' (referred to as "restricted ethyl" or "cEt" in the S configuration), 4'-CH2-O-CH2-2', and 4'-C H2-N(R)-2', 4'-CH(CH2OCH3)-O-2' ("constrained MOE" or "cMOE") and its analogues, 4'-C(CH3)(CH3)-O-2' and its analogues, 4'-CH2-N(OCH3)-2' and its analogues, 4'-CH2-ON(CH3)-2', 4'-CH2-C(H)(CH3)-2', 4'-CH2-C(=CH2)-2' and its analogues, 4'-C(R a R b )-N(R)-O-2',4'-C(R a R b )-ON(R)-2', 4'-CH2-ON(R)-2', and 4'-CH2-N(R)-O-2' (wherein each R, R a , and R b These are independently H, protecting group, or C1-C 12Examples include, but are not limited to, alkyl groups. Representative U.S. patents teaching the preparation of such bicyclic sugar moieties include, but are not limited to, US7,427,672 by Imanishi et al., US7,741,457 by Swayze et al., US8,022,193 by Swayze et al., US8,278,283 by Seth et al., US8,278,425 by Prakash et al., and US8,278,426 by Seth et al.

[0414] 2. Certain modified nucleic acid bases In certain embodiments, a modified oligonucleotide comprises one or more nucleosides containing unmodified nucleic acid bases. In certain embodiments, a modified oligonucleotide comprises one or more nucleosides containing modified nucleic acid bases. In certain embodiments, a modified oligonucleotide comprises one or more nucleosides that do not contain nucleic acid bases, referred to as debased nucleosides. In certain embodiments, a modified oligonucleotide comprises one or more inosine nucleosides (i.e., nucleosides containing hypoxanthine nucleic acid bases). An "unmodified nucleic acid base" is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). A "modified nucleic acid base" is an atomic group other than unmodified A, T, C, U, or G that can pair with at least one other nucleic acid base. 5-methylcytosine is an example of a modified nucleic acid base. A universal base is a modified nucleic acid base that can pair with any one of the five unmodified nucleic acid bases.

[0415] In certain embodiments, the modified nucleic acid base is selected from 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl-substituted pyrimidines, alkyl-substituted purines, and N-2, N-6, and O-6-substituted purines. In certain embodiments, the modified nucleic acid bases are 5-methylcytosine, 2-aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-propynyl(-C≡C-CH3)uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azocymine, 5-ribosyluracil (pseudracil), N1-methylpseudracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8- Aza and other 8-substituted purines, 5-halo (especially 5-bromo), 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl4-N-benzoylcytosine, 5-methyl4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases are selected. Further modified nucleic acid bases include tricyclic pyrimidines such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleic acid bases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Further nucleic acid bases include those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke ST, Ed., CRC Press, 2008, 163-166 and 442-443.

[0416] Publications that teach the preparation of certain modified nucleic acid bases among the above-mentioned modified nucleic acid bases, as well as other modified nucleic acid bases, include, but are not limited to, US5,134,066 by Rogers et al., US5,432,272 by Benner et al., US5,502,177 by Matteucci et al., US5,594,121 by Froehler et al., and US5,681,941 by Cook et al.

[0417] B. Certain modified nucleoside interbonding The naturally occurring nucleoside-to-nucleoside bonds in RNA and DNA are 3'-to-5' phosphodiester bonds. In certain embodiments, nucleosides of modified oligonucleotides may be linked to one another using one or more modified nucleoside-to-nucleoside bonds. Two main classes of nucleoside-to-nucleoside bonds are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing nucleoside-to-nucleoside bonds include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, phosphorothioates, phosphonoacetates ("PACE"), thiophosphonoacetates ("Thio-PACE"), and phosphorodithioates, which include phosphodiester bonds (also referred to as unmodified or naturally occurring bonds). Representative phosphorus-free nucleoside interbonding groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters, thionocarbamates (-OC(=O)(NH)-S-), siloxanes (-O-SiH2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified nucleoside interbonding groups can be used to modify, typically increase, the nuclease resistance of oligonucleotides compared to naturally occurring phosphodiester nucleoside interbonding groups. In certain embodiments, nucleoside interbonding groups having chiral atoms can be prepared as racemic mixtures or as separate enantiomers. Methods for preparing phosphorus-containing and phosphorus-free nucleoside interbonding groups are well known to those skilled in the art.

[0418] In certain embodiments, the modified nucleoside bond is one of those described in WO2021 / 030778 (incorporated herein by reference). In certain embodiments, the modified nucleoside bond is of formula: [ka] The formula includes, and for each nucleoside-bonding group of the modified oligonucleotide, independently, X is selected from O or S. R1 is selected from H, C1-C6 alkyl, and substituted C1-C6 alkyl. T is selected from SO2R2, C(=O)R3, and P(=O)R4R5. R2 is selected from aryl, substituted aryl, heterocycle, substituted heterocycle, aromatic heterocycle, substituted aromatic heterocycle, diazole, substituted diazole, C1-C6 alkoxy, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, substituted C1-C6 alkyl, substituted C1-C6 alkenyl-substituted C1-C6 alkynyl, and linker. R3 is selected from aryl, substituted aryl, CH3, N(CH3)2, OCH3, and linker. R4 is selected from OCH3, OH, C1-C6 alkyl, substituted C1-C6 alkyl, and linker. R5 is selected from OCH3, OH, C1-C6 alkyl, and substituted C1-C6 alkyl.

[0419] In certain embodiments, the modified nucleoside bond includes a mesylphosphoamide bond having the following formula: [ka] .

[0420] In certain embodiments, the modified nucleoside bond is a neutral nucleoside bond. Neutral nucleoside bonds include, but are not limited to, phosphotriesters, methylphosphonates, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), methoxypropyl (MOP), and thioformacetal (3'-S-CH2-O-5'). Further neutral nucleoside bonds include nonionic bonds involving siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters, and amides (see, for example, Carbohydrate Modifications in Antisense Research; YSSanghvi and PDCook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral nucleoside bonds include nonionic bonds involving mixed N, O, S, and CH2 component moieties.

[0421] In certain embodiments, the modified nucleoside bond is a standard-length nucleoside bond, represented by the following formulas Z1, Z2, or Z3. As used herein, “standard-length nucleoside bond” means a nucleoside bond having the structure represented by formulas Z1, Z2, or Z3. [ka] For each nucleoside bond group in formula Z1, Z2, or Z3, independently, each X 1 These are independently selected from O and S, X 2 O, NR 1 Selected from CH2 and S, X 3 O, NR 1 Selected from CH2 and S, L is absent, NR 1, N(R 1 )SO2, -N=, O, C1-C6 alkylene, or C1-C6 heteroalkylene, Each R 1 These are independently selected from H, C1-C6 alkyl, and substituted C1-C6 alkyl, or two R on the same atom. 1 However, together they form =O, R 2 -OH, -SH, C1-C 22 Alkyl, substituted C1-C 22 Alkyl, C2-C 22 Alkenyl, substituted C2-C 22 Selected from alkenyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, heteroaryl, substituted heteroaryl, aryl, and substituted aryl, If the group is substituted, the group is -OH, -N(R) 1 )2,-O-C1-C6 alkyl, C1-C 22 Alkyl, C2-C 22 It comprises one or more substituents selected from alkenyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl.

[0422] In certain embodiments, an extended nucleoside bond is an extended-length nucleoside bond. For example, in certain embodiments, an extended nucleoside bond is formed when two functional groups on two oligonucleotides react to link the two oligonucleotides together to form a single oligonucleotide containing an extended nucleoside bond. One such reaction is the click reaction between bicyclo[6.1.0]nonine and azide. Additional linkers suitable for linking two oligonucleotides in click chemistry are described in "Click Chemistry for Biotechnology and Materials Science" (Ed. Joerg Lahann, Wiley 2009). Further examples of bonding chemistry include, for example, the reverse electron-required Diels-Alder reaction (e.g., Argamunt et al., J.Org.Chem.2020,85,10,6593-6604, Sarrett et al., Nat.Protocols 2021,16,3348-3381, Handula et al., Molecules,2021,26(15),4640, Wiessler et al., Int.J.Med.Sci.2010,7(1),19-28), and copper-catalyzed azide-alkyne cycloaddition (CuAAC) (e.g., SIPresolski, et al., J.Am.Chem.Soc.2010,132,14570-14576, D.Soriano Del Amo, et al.) See al., J. Am. Chem. Soc., 2010, 132, 16893-16899, the Staudinger reaction (e.g., see Saxon and CRBertozzi, Science, 2000, 287, 2007-2010, BL Nilsson, et al., Org. Lett., 2000, 2, 1939-1941, E. Saxon, et al., Org. Lett., 2000, 2, 2141-2143), the formation of hydrazones and oximes (e.g., see JYAxup, et al., Proc. Natl. Acad. Sci. USA, 2012, 109, 16101-16106), and the photoclick reaction (e.g., W. Song, et al., Angew. Chem., Int. Ed.See A. Herner and Q. Lin, Top. Curr. Chem., 2016, 374, 1), strain-promoted alkyne-nitrone cycloaddition (SPANC) reactions (e.g., see DAM Mac Kenzie, et al., Curr. Opin. Chem. Biol., 2014, 21, 81-88), transition metal-catalyzed cross-coupling (e.g., see M. Chalker, et al., J. Am. Chem. Soc., 2009, 131, 16346-16347), nucleophilic addition (e.g., addition of thiols to maleimides) (e.g., see Kang et al., Chem. Sci., 2021, 12, 13613-13647, Bernardim et al.) (See also al., Nat.Comm. 2016, 7, 13128 and Jain et al., Pharm.Res. 2015, 32(11), 3526-3540).

[0423] C. A specific motif In certain embodiments, the guide (modified oligonucleotide) comprises one or more modified nucleosides containing a modified sugar moiety. In certain embodiments, the modified oligonucleotide comprises one or more modified nucleosides containing a modified nucleic acid base. In certain embodiments, the modified oligonucleotide comprises one or more modified internucleoside bonds. In such embodiments, the modified, unmodified, and different modified sugar moieties, nucleic acid bases, and / or internucleoside bonds of the modified oligonucleotide define a certain pattern or motif. In certain embodiments, the patterns of sugar moieties, nucleic acid bases, and internucleoside bonds are each independent of each other. Thus, the modified oligonucleotide can be described by its sugar motif, nucleic acid base motif, and / or internucleoside bond motif (wherein use herein, the nucleic acid base motif indicates a modification to the nucleic acid base, independently of the sequence of the nucleic acid base).

[0424] 1. A specific sugar motif In certain embodiments, the oligonucleotide comprises one or more modified sugars and / or unmodified sugar moieties arranged along the oligonucleotide or region within a defined pattern or sugar motif. In certain examples, such sugar motifs include, but are not limited to, any of the sugar modifications considered herein. Generally, the guide comprises an unmodified RNA nucleoside and, optionally, one or more modified nucleosides described herein. In certain embodiments, the guide comprises or consists of a modified oligonucleotide.

[0425] In certain embodiments, the 5'-side nucleoside of the modified oligonucleotide contains a modified sugar moiety. In certain embodiments, the 3'-side nucleoside of the modified oligonucleotide contains a modified sugar moiety. In certain embodiments, at least one, two, three, four, or five of the five 5'-side nucleosides of the modified oligonucleotide contain a modified sugar moiety. In certain embodiments, at least one, two, three, four, or five of the five 3'-side nucleosides of the modified oligonucleotide contain a modified sugar moiety.

[0426] In certain embodiments, the above modifications (sugars, nucleic acid bases, nucleoside bonds) are incorporated into the modified oligonucleotide. In certain embodiments, the modified oligonucleotide is characterized by its modification motifs and full length as described herein, and such parameters are independent of each other. Unless otherwise indicated, each modification is independent of the nucleic acid base sequence. In certain embodiments, the modified oligonucleotide is a guide. In certain embodiments, the guide has a sugar moiety, nucleic acid base, and / or nucleoside-linking motif described in any of the following references: WO2014 / 144761, WO2015 / 026885, WO2016 / 089433, WO2016 / 100951, WO2016 / 123230, WO2016 / 164356, WO2017 / 004261, WO2017 / 004279, WO2017 / 068377, WO2017 / 136794, WO2017 / 181107, WO2017 / 214460, WO20 18 / 009822, WO2018 / 057946, WO2018 / 098383, WO2018 / 107028, WO2018 / 125964, WO2019 / 084664, WO2019 / 147275, WO2019 / 147743, WO2019 / 183000, WO2019 / 237069, WO2021 / 119006, WO2021 / 119275, WO2021 / 125840, WO2021 / 207651, WO2021 / 207711, WO2022 / 086846 (each of these is incorporated herein by reference).

[0427] 2. A specific nucleic acid base motif In certain embodiments, the oligonucleotide comprises modified and / or unmodified nucleic acid bases arranged along the oligonucleotide or region within a defined pattern or motif. In certain embodiments, each nucleic acid base is modified. In certain embodiments, none of the nucleic acid bases are modified. In certain embodiments, each purine or pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleic acid bases in the modified oligonucleotide are 5-methylcytosine. In certain embodiments, all of the cytosine nucleic acid bases are 5-methylcytosine, and all of the other nucleic acid bases in the modified oligonucleotide are unmodified nucleic acid bases.

[0428] In certain embodiments, the modified oligonucleotide includes a block of modified nucleic acid bases. In certain such embodiments, the block is located at the 3' end of the oligonucleotide. In certain embodiments, the block is located within 3 nucleosides from the 3' end of the oligonucleotide. In certain embodiments, the block is located at the 5' end of the oligonucleotide. In certain embodiments, the block is located within 3 nucleosides from the 5' end of the oligonucleotide.

[0429] In certain embodiments, the modified nucleic acid base is selected from pseudouracil, 2-6-diaminopurine, 2-thiouracil, 4-thiouracil, 2-aminoadenine, 6-methyladenine, hypoxanthine, or 5-methylcytosine.

[0430] 3. A specific nucleoside bond motif In certain embodiments, the oligonucleotide comprises modified and / or unmodified nucleoside bonds arranged along the oligonucleotide or its region in a defined pattern or motif. In certain embodiments, each nucleoside bond group is a phosphodiester nucleoside bond (P=O). In certain embodiments, each nucleoside bond group of the modified oligonucleotide is a phosphorothioate nucleoside bond (P=S). In certain embodiments, each nucleoside bond of the modified oligonucleotide is independently selected from phosphorothioate nucleoside bonds and phosphodiester nucleoside bonds. In certain embodiments, each nucleoside bond of the modified oligonucleotide is independently selected from phosphorothioate nucleoside bonds, phosphodiester nucleoside bonds, and phosphonoacetate nucleoside bonds.

[0431] In certain embodiments, the 5'-side nucleoside bond of the modified oligonucleotide is a modified nucleoside bond. In certain embodiments, the 3'-side nucleoside bond of the guide is a modified nucleoside bond. In certain embodiments, at least one, two, three, four, or five of the five 5'-side nucleoside bonds of the modified oligonucleotide are modified nucleoside bonds. In certain embodiments, at least one, two, three, four, or five of the five 3'-side nucleoside bonds of the modified oligonucleotide are modified nucleoside bonds. In certain embodiments, the modified nucleoside bond is a phosphorothioate nucleoside bond.

[0432] 4. A specific guide chemical modification motif The guide may include any of the modifications commonly applied to modified oligonucleotides described herein, including modified sugar moieties, modified nucleoside-to-nucleoside bonds, and modified nucleic acid bases. In certain embodiments, the guide is modified at the 3' end, the 5' end, or both the 3' and 5' ends. In certain embodiments, three nucleosides at the 3' end and three nucleosides at the 5' end are 2'-OMe nucleosides, and the remainder of the guide nucleosides are unmodified RNA nucleosides. In certain embodiments, each nucleoside in the most 5'-side stem loop is an unmodified RNA nucleoside. In certain embodiments,

[0433] In certain embodiments, each nucleoside bond in the guide is an unmodified phosphodiester bond. In certain embodiments, the guide includes one or more modified nucleoside bonds. In certain embodiments, the guide includes one or more phosphorothioate nucleoside bonds. In certain embodiments, the 1st, 2nd, 3rd, 4th, or 5th nucleoside bonds on the 3' end are modified nucleoside bonds. In certain embodiments, the 1st, 2nd, 3rd, 4th, or 5th nucleoside bonds on the 5' end are modified nucleoside bonds. In certain embodiments, the 1st, 2nd, 3rd, 4th, or 5th nucleoside bonds on the 3' end and the 5' end are modified nucleoside bonds, and the remainder of the bonds are unmodified phosphodiester nucleoside bonds. In certain embodiments, the 1st, 2nd, 3rd, 4th, or 5th nucleoside bonds on the 3' end are phosphorothioate nucleoside bonds. In certain embodiments, the 1st, 2nd, 3rd, 4th, or 5th nucleoside bonds on the 5' end are phosphorothioate nucleoside bonds. In certain embodiments, the 1st, 2nd, 3rd, 4th, or 5th nucleoside bonds on the 3' end and the 5' end are phosphorothioate nucleoside bonds, and the remainder of the bonds are unmodified phosphodiester nucleoside bonds. In certain embodiments, three of the 3' end and three of the 5' end nucleoside bonds are phosphorothioate nucleoside bonds, and the remainder of the bonds are unmodified phosphodiester nucleoside bonds.

[0434] D. A certain length In certain embodiments, the oligonucleotide (including the guide) can have any of a diverse range of lengths. In certain embodiments, the oligonucleotide consists of X to Y bound nucleosides, where X represents the minimum number of nucleosides in the range and Y represents the maximum number of nucleosides in the range. In certain such embodiments, X and Y are independently 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112 ,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,1 Select from 49, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, provided that X ≤ Y. For example, in a particular embodiment, the oligonucleotide consists of 50-180, 80-180, 90-180, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 160-170, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 100-140, 110-140, 110-130, 120-140, or 120-150 linked nucleosides.

[0435] In a particular embodiment, the single guide oligonucleotide consists of 50-180, 80-180, 90-180, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 160-170, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 100-140, 110-140, 110-130, 120-140, or 120-150 conjugated nucleosides. In a particular embodiment, the target recognition region of the guide consists of 15-30, 18-26, 18-24, 18-22, 20-26, 20-24, 20-22, 21-23, 22-23, 20, 21, 22, or 23 bound nucleosides.

[0436] In a particular embodiment, the protein recognition region of the guide is 60-130, 60-120, 60-110, 60-100, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 80-130, 80-120, 80-110, 80-100, 80-90, 90-130, 90-120, 90-110 It consists of 90-100, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 bound nucleosides. In certain embodiments, the protein recognition region of the guide represents a cleaved version of the native guide of the corresponding Cas protein. In certain embodiments, the guide is cleaved at its 3' end. In certain embodiments, the guide is shortened by removing nucleotides from its stem-loop and / or hairpin structure (e.g., shortening the hairpin by removing one base pair from it). In certain embodiments, the cleaved version is at least 5, at least 10, at least 15, or at least 20 nucleotides shorter than the natural guide. In certain embodiments, the protein recognition region of the guide represents an extended version of the natural guide of the corresponding Cas protein. In certain embodiments, additional nucleotides are appended to the 3' end. In certain embodiments, additional nucleotides are inserted into the loops and / or hairpins of the natural guide.

[0437] E. Nucleic acid base sequence In certain embodiments, oligonucleotides (unmodified or modified oligonucleotides) are further described by their nucleic acid base sequences. In certain embodiments, the oligonucleotide has a nucleic acid base sequence complementary to a specified reference nucleic acid, such as a second oligonucleotide or target nucleic acid. In certain such embodiments, a region of the oligonucleotide has a nucleic acid base sequence complementary to a specified reference nucleic acid, such as a second oligonucleotide or target nucleic acid. In certain embodiments, a region or the entire nucleic acid base sequence of the oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a nucleic acid such as a second oligonucleotide or target nucleic acid.

[0438] F. Secondary structure In certain embodiments, the oligonucleotide or a portion thereof takes on a defined secondary structure. In certain embodiments, the secondary structure is determined by Watson-Crick base pairing, Hoogsteen base pairing, and / or non-canonical base pairing interactions. The secondary structure of an oligonucleotide sequence can be predicted using standard software such as the Vienna RNA package RNAfold (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi; see Lorenz, et al., Algorithms for Molecular Biology, 6:1 26, 2011). In certain embodiments, the oligonucleotide may have two or more predicted secondary structures.

[0439] Guide oligonucleotides possess a secondary structure, at least a portion of which is crucial for forming contact with their congener Cas proteins. For example, the secondary structural features of guides for binding to spCas9 have been described in detail based on both empirical evidence (see, e.g., Zhang, et al., ChemPlusChem, 2021 and Dong, et al., Curr. Opinion in Biotech., 2022) and the crystal structure of the guide-SpCas9-target DNA complex (see Nishimasu, et al., Cell, 2014). By adding a tetraloop to the double helix formed between the native crRNA and tracrRNA of SpCas9, the functions of the crRNA and tracrRNA are combined into a single guide RNA. The same tetraloop can be added to ligate the crRNA and tracrRNA of novel type II Cas proteins. The single guide of SpCas9 has conserved structural features within its protein-binding region, including a lower stem, upper stem, tetraloop, bulge, nexus region (a hairpin with a 5-nucleotide loop), and two hairpins at the 3' end (Figure 6). Guide RNAs of certain novel type II Cas proteins have been shown to have unique secondary structures with common features in their protein-binding region, including a double-stranded region (dual guide) or stem-loop (single guide) and one or more hairpins at the 3' end. The feature of the stem-loop on the 5' end of the guide RNA is also known as the repeat-anti-repeat region (R-AR).

[0440] In a particular embodiment, the guide consists of oligonucleotides that can be represented by the following formula: T1-DXS 1a -B1-S 1b -H1-S 1b '-B2-S 1a '-L1-S2-H2-S2'-L2-S3-H3-S3'-(L3-S4-H4-S4') n -(L4-S5-H5-S5') m -T2, In the array, D consists of 15 to 25 linked nucleosides, and the nucleic acid base sequence of D is complementary to the nucleic acid base sequence of the target DNA. X consists of one or two bound nucleosides that are either absent or not complementary to the target DNA sequence. Each of T1 and T2 is either absent or independently consists of 1 to 30 bound nucleosides. S 1a and S 1a Each of these consists of 6 to 12 bonded nucleosides, S 1a The nucleic acid base sequence is S 1a It is 100% complementary to the nucleic acid base sequence of ', S 1b and S 1b Each of these consists of 2 to 14 bonded nucleosides, S 1b The nucleic acid base sequence is S 1b It is 100% complementary to the nucleic acid base sequence of ', S2 and S2' each consist of 2 to 6 bonded nucleosides, and the nucleic acid base sequence of S2 is 100% complementary to the nucleic acid base sequence of S2'. S3 and S3' each consist of 2 to 12 bonded nucleosides, and the nucleic acid base sequence of S3 is 100% complementary to the nucleic acid base sequence of S3'. S4 and S4' each consist of 4 to 14 bonded nucleosides, and the nucleic acid base sequence of S4 is 100% complementary to the nucleic acid base sequence of S4'. S5 and S5' each consist of 4 to 11 bonded nucleosides, and the nucleic acid base sequence of S5 is 100% complementary to the nucleic acid base sequence of S5'. n is 0 or 1, m is 0 or 1, B1 consists of 0 to 3 bonded nucleosides. B2 consists of 0 to 4 bonded nucleosides. Except when both B1 and B2 consist of zero bonded nucleosides, B1 is not equal to B2. Each of H1, H2, H3, H4, and H5 independently consists of 3 to 6 bonded nucleosides. L1 consists of 2-3 bonded nucleosides, Each of L2, L3, and L4 independently consists of 0 to 10 bonded nucleosides.

[0441] In a particular embodiment, the guide specific to the ION-CAS protein is represented by the following formula: T1-DXS 1a -B1-S 1b -H1-S 1b '-B2-S 1a '-L1-S2-H2-S2'-L2-S3-H3-S3'-(L3-S4-H4-S4') n -(L4-S5-H5-S5') m -T2, Within the array, each variable is defined as shown in the table below. [Table 4]

[0442] The secondary structural features of guides involved in binding to Cas enzymes include having at least one double-chain region at the 5' end of the protein recognition region (including one or more stem-loops in the case of single guides), as well as two or more additional stem-loops and / or hairpin regions. For most type II Cas proteins, the bulge at the 5' end of the stem-loop is conserved among guides, although guides with a variety of predicted secondary structures have also been reported (see, e.g., Alexander et al., CRISPR J., 6(3):261-277, 2023).

[0443] In certain embodiments, the activity of the guide is driven not by a specific nucleic acid base sequence, but by the formation of a specific RNA secondary structure. The sequence that forms this secondary structure can be modified. One common method of modifying the sequence while maintaining the three-dimensional structure necessary for the guide to bind to its homologous Cas is to flip base pairs within the stem or hairpin, such as an "AU" flip. In such embodiments, the native AU base pair is replaced with a UA base pair, swapping the positions of "A" and "U" in the guide sequence. This can particularly improve editing efficiency if the original guide contains four consecutive Us that are putative pol-III terminators (e.g., Chen et al. Cell. (2013); 155(7): 1479-1491). Another method of modifying the sequence within the stem or hairpin involves replacing less stable AU or UA base pairs with more stable CG or GC base pairs to increase overall hairpin stability (see, for example, Riesenberg, et al., Nat. Commun., 2022).

[0444] In certain embodiments, the native hairpin or stem-loop structure of the guide molecule is extended or replaced by an extended stem-loop. In certain cases, it has been demonstrated that stem extension can enhance the assembly of the guide with the CRISPR-Cas protein (Chen et al. Cell. (2013); 155(7): 1479-1491). In certain embodiments, the stem of the stem-loop is extended by at least 1, 2, 3, 4, 5, or more complementary base pairs (i.e., corresponding to the addition of 2, 4, 6, 8, 10, or more nucleotides in the guide). In certain embodiments, these are located at the ends of the stem, adjacent to the loop of the stem-loop. In certain embodiments, the native hairpin or stem-loop structure of the guide is shortened. In certain such embodiments, the stem of the stem-loop is shortened by only 1, 2, or 3 complementary base pairs (i.e., corresponding to the removal of 2, 4, or 6 nucleotides in the guide).

[0445] Furthermore, in the case of SpCas9 guides, it has been demonstrated that the loop region of the upper stem or hairpin 1 can be replaced by a longer loop containing an aptamer. In certain embodiments, the aptamer binds to the effector protein. In certain embodiments, the aptamer binds to MS2, PP7, com, or box B. In certain embodiments, the aptamer can be attached to the 3' end of the guide, or it can be inserted into the guide as a new hairpin beyond the most 3' hairpin of the natural guide (see, for example, Zalatan, et al, Cell, 2015).

[0446] In certain embodiments, the guide can be modified to be activated only by an external chemical signal. In one such embodiment, the guide includes a 5' extension attached to the 5' end of the target recognition region of the guide, which forms a hairpin double helix with the target recognition region. This 5' extension also includes a theophylline aptamer. In this system, the addition of theophylline causes cleavage of the 5' extension, exposing the target recognition region of the guide. In certain embodiments, the guide can be modified to be deactivated by an external chemical signal. In one such embodiment, the guide includes a theophylline aptamer incorporated into the loop region of a first stem-loop and a second theophylline aptamer incorporated into the loop of a second hairpin. This guide functions for Cas9 cleavage of the target but is cleaved when theophylline is added to the system. Various other external control systems for guides have been described (see, for example, Zhang, et al., ChemPlusChem, 2021).

[0447] C. Guide Chemical Modification The guide may include any of the modifications commonly applied to modified oligonucleotides described herein, including modified sugar moieties, modified nucleoside-to-nucleoside bonds, and modified nucleic acid bases. In certain embodiments, the guide is modified at the 3' end, the 5' end, or both the 3' and 5' ends. In certain embodiments, three nucleosides at the 3' end and three nucleosides at the 5' end are 2'-OMe nucleosides, and the remainder of the guide nucleosides are unmodified RNA nucleosides. In certain embodiments, each nucleoside in the most 5'-side stem loop is an unmodified RNA nucleoside. In certain embodiments,

[0448] In certain embodiments, each nucleoside bond in the guide is an unmodified phosphodiester bond. In certain embodiments, the guide includes one or more modified nucleoside bonds. In certain embodiments, the guide includes one or more phosphorothioate nucleoside bonds. In certain embodiments, the 1st, 2nd, 3rd, 4th, or 5th nucleoside bonds on the 3' end are modified nucleoside bonds. In certain embodiments, the 1st, 2nd, 3rd, 4th, or 5th nucleoside bonds on the 5' end are modified nucleoside bonds. In certain embodiments, the 1st, 2nd, 3rd, 4th, or 5th nucleoside bonds on the 3' end and the 5' end are modified nucleoside bonds, and the remainder of the bonds are unmodified phosphodiester nucleoside bonds. In certain embodiments, the 1st, 2nd, 3rd, 4th, or 5th nucleoside bonds on the 3' end are phosphorothioate nucleoside bonds. In certain embodiments, the 1st, 2nd, 3rd, 4th, or 5th nucleoside bonds on the 5' end are phosphorothioate nucleoside bonds. In certain embodiments, the 1st, 2nd, 3rd, 4th, or 5th nucleoside bonds on the 3' end and the 5' end are phosphorothioate nucleoside bonds, and the remainder of the bonds are unmodified phosphodiester nucleoside bonds. In certain embodiments, three of the 3' end and three of the 5' end nucleoside bonds are phosphorothioate nucleoside bonds, and the remainder of the bonds are unmodified phosphodiester nucleoside bonds.

[0449] III. A specific editing system In certain embodiments, the editing system includes a guide nucleic acid binder and a guide. In certain embodiments, the guide consists of a single oligonucleotide and is a single guide. In certain embodiments, the guide consists of two oligonucleotides and is a dual guide. In certain embodiments, the guide includes a target recognition region and a protein recognition region. In certain embodiments, the guide is a single guide consisting of oligonucleotides comprising the target recognition region and the protein recognition region. In certain embodiments, the guide is a dual guide, where the first oligonucleotide comprises the first components of the target recognition region and the protein recognition region, and the second oligonucleotide comprises the second component of the protein recognition region. In certain such embodiments, the first oligonucleotide is "crRNA" and the second oligonucleotide is "tracrRNA". In certain embodiments, the target recognition region is a region of oligonucleotide having a sequence complementary to an isolength portion of the target sequence in the complementary strand of the target DNA.

[0450] In certain embodiments, the editing system includes a guide nucleic acid binder and a guide. In certain embodiments, the guide nucleic acid binder includes a Cas protein. In certain embodiments, the guide nucleic acid binder consists of a Cas protein. In certain embodiments, the Cas protein has one of the amino acid sequences of SEQ ID NOs: 4-23 or 231-323. In certain embodiments, the Cas protein is encoded by DNA having one of the nucleic acid base sequences of SEQ ID NOs: 24-43 or 324-416. In certain embodiments, the guide is an oligonucleotide. In certain embodiments, the guide consists of two oligonucleotides. In certain embodiments, the guide includes a protein recognition region having a nucleic acid base sequence selected from SEQ ID NOs: 121-141, 510-602, 961-1408, 2825, or 2827-2833. In certain embodiments, the editing system includes a guide nucleic acid binder containing a guide nucleic acid protein and a guide, wherein the protein recognition region of the guide has a nucleic acid base sequence that binds to the guide nucleic acid protein. In certain embodiments, the guide nucleic acid binder includes an ION-Cas protein. In a particular embodiment, the editing system includes an ION-Cas protein having the amino acid sequence of SEQ ID NO: 12 and a guide including a protein recognition region having the nucleic acid sequence of SEQ ID NO: 129 or 130. In a particular embodiment, the editing system includes an ION-Cas protein having the amino acid sequence of SEQ ID NO: 13 and a guide including a protein recognition region having the nucleic acid sequences of SEQ ID NO: 131, 961-964, 1016-1020, 1052-1070, 1313-1314, or 2825. In a particular embodiment, the editing system includes an ION-Cas protein having the amino acid sequence of SEQ ID NO: 20 and a guide including a protein recognition region having the nucleic acid sequence of SEQ ID NO: 139. In a particular embodiment, the editing system includes an ION-Cas protein having the amino acid sequence of SEQ ID NO: 241 and a guide including a protein recognition region having the nucleic acid sequences of SEQ ID NO: 520, 965-968, 1071-1094, or 1315-1318.In a particular embodiment, the editing system includes an ION-Cas protein having the amino acid sequence of SEQ ID NO: 267 and a guide including a protein recognition region having the nucleic acid sequences of SEQ ID NO: 546, 973-976, 1021-1023, 1095-1126, or 1319-1342. In a particular embodiment, the editing system includes an ION-Cas protein having the amino acid sequence of SEQ ID NO: 275 and a guide including a protein recognition region having the nucleic acid sequences of SEQ ID NO: 554, 981-984, 1024-1026, 1127-1152, or 1343-1361. In a particular embodiment, the editing system includes an ION-Cas protein having the amino acid sequence of SEQ ID NO: 284 and a guide including a protein recognition region having the nucleic acid sequences of SEQ ID NO: 562, 985-989, 1027-1029, 1153-1182, or 1362-1390. In a particular embodiment, the editing system includes an ION-Cas protein having the amino acid sequence of SEQ ID NO: 289 and a guide including a protein recognition region having the nucleic acid sequences of SEQ ID NO: 568, 990-993, 1030-1034, 1183-1206, or 1392-1393. In a particular embodiment, the editing system includes an ION-Cas protein having the amino acid sequence of SEQ ID NO: 314 and a guide including a protein recognition region having the nucleic acid sequences of SEQ ID NO: 593, 1006-1010, 1272-1292, 1394-1408, or 2827-2833.

[0451] In a particular embodiment, the editing system includes an ION-Cas protein having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12, and a guide including a protein recognition region having a nucleic acid base sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 129 or 130. In a particular embodiment, the editing system includes an ION-Cas protein having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13, and a guide having a protein recognition region having a nucleic acid base sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 131, 961-964, 1016-1020, 1052-1070, 1313-1314, or 2825. In a particular embodiment, the editing system includes an ION-Cas protein having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20, and a guide including a protein recognition region having a nucleic acid base sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 141.

[0452] In a particular embodiment, the editing system includes an ION-Cas protein having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 241, and a guide having a protein recognition region having a nucleic acid base sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 520, 965-968, 1071-1094, or 1315-1318. In a particular embodiment, the editing system includes an ION-Cas protein having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 267, and a guide having a protein recognition region having a nucleic acid base sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 546, 973-976, 1021-1023, 1095-1126, or 1319-1342. In a particular embodiment, the editing system includes an ION-Cas protein having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 275, and a guide having a protein recognition region having a nucleic acid base sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 554, 981-984, 1024-1026, 1127-1152, or 1343-1361.In a particular embodiment, the editing system includes an ION-Cas protein having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 284, and a guide having a protein recognition region having a nucleic acid base sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 562, 985-989, 1027-1029, 1153-1182, or 1362-1390. In a particular embodiment, the editing system includes an ION-Cas protein having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 289, and a guide having a protein recognition region having a nucleic acid base sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 568, 990-993, 1030-1034, 1183-1206, or 1392-1393. In a particular embodiment, the editing system includes an ION-Cas protein having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 314, and a guide having a protein recognition region having a nucleic acid base sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 593, 1006-1010, 1272-1292, 1394-1408, or 2827-2833.

[0453] IV. A specific delivery vehicle A. Lipid nanoparticles ("LNPs") 1. Composition In certain embodiments, the LNP comprises a protonable or cationic lipid, a neutral or amphoteric lipid, a polymeric lipid, cholesterol or a derivative thereof, and optionally one or more excipients. The LNP at least partially encapsulates the cargo. In certain embodiments, the cargo comprises a nucleic acid (e.g., exogenous mRNA). In certain embodiments, the cargo comprises a guide (e.g., an oligomeric agent). In certain embodiments, the cargo comprises exogenous mRNA and a guide. In certain embodiments, the LNP is provided as a suspension in an aqueous medium. In certain embodiments, the pharmaceutical composition comprises an LNP that at least partially encapsulates the cargo in an aqueous medium.

[0454] In certain embodiments, the LNP comprises 30–70 mol% protonable or cationic lipids, 5–30 mol% neutral or amphoteric lipids, 20–50 mol% cholesterol or its derivatives, and 1–10 mol% polymeric lipids. In certain embodiments, the LNP comprises 40–60 mol% protonable or cationic lipids, 5–15 mol% neutral or amphoteric lipids, 30–50 mol% cholesterol or its derivatives, and 2–4 mol% polymeric lipids. The mol% of each of the protonable or cationic lipids, neutral or amphoteric lipids, polymeric lipids, cholesterol, or its derivatives is determined together regardless of any cargo or excipients ("total lipids").

[0455] LNPs can be characterized by the molar or mass ratio of cargo (e.g., exogenous mRNA or oligomeric agent) to total lipids. In certain embodiments, the lipid-to-cargo ratio is 20:1–1:1, 10:1–1:1, or 5:1–1:1 (mass:mass).

[0456] The LNP composition may contain one or more additional excipients, such as hydrophobic excipients including triglycerides, surfactants, and / or waxes. Triglycerides include trimiristine (Dynasan 114), tripalmitine (Dynasan 116), or tristearin (Dynasan 118), vitepsol bases, glyceryl stearate (Imwitor 900), glyceryl behenate (Compritol 888 ATO), and glyceryl palmitostearate (Precirol ATO 5). Nonionic surfactants may include moieties such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters, nonionic alkanolamides, and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers. Anionic surfactants include carboxylates, acyl lactylates, acylamides of amino acids, sulfuric acid esters (e.g., alkyl sulfates and ethoxylated alkyl sulfates), sulfonates (e.g., alkylbenzene sulfonates), acyl isethionates, acyl taurates, and sulfosuccinates, as well as phosphates. Specific surfactants include lecithin, Poloxamer 188, Poloxamer 407, Tyloxapol, Polysorbate 20, Polysorbate 60, Polysorbate 80, sodium cholate, sodium glycocholate, sodium taurodeoxycholate, butanol, butyric acid, cetylpyridinium chloride, sodium dodecyl sulfate, sodium oleate, polyvinyl alcohol, or Cremophor EL. Waxes include beeswax and cetyl palmitate. Other hydrophobic excipients include stearic acid, palmitic acid, behenic acid, Miglyol 812, and paraffin.

[0457] a. Protonable lipids and cationic lipids In certain embodiments, LNPs comprising protonable and / or cationic lipids are provided. As used herein, “protonable lipid” refers to a lipid-containing compound that is substantially protonated below a physiological pH (e.g., pH 7–7.5, or pH 7.4). As used herein, “cationic lipid” refers to a lipid-containing compound that carries an irreplaceable net positive charge. Lipids generally consist of a hydrophobic moiety that may include hydrocarbon chains.

[0458] The design of protonable and / or cationic lipids is thought to enhance endosomal drug escape. Therefore, in vivo, the delivery capability of LNPs is thought to benefit from electrostatic interactions with endosomes and subsequent destabilization of LNPs, which lead to cargo release. Protonable lipids are thought to aid delivery by (i) protonating and acquiring a positive charge in a mildly acidic environment, and / or (ii) incorporating pH-unstable groups that are cleaved or conformationally altered in a low pH environment. Thus, such pH-sensitive lipids help induce cargo release by enhancing charge-based uptake by liposomes in target cells or by destabilizing the liposome membrane. Cationic lipids can be characterized by an inverted cubic or hexagonal liquid crystal phase.

[0459] Fusibility is a property that describes the ability of lipid or polylipid constructs to fuse with lipid layers. See, for example, Hashiba et al., Small Science, 3(1), 2023, 2200071 and V. Gyanani, and R. Goswami, Pharmaceuticals, 2023, 15(4):1184. Lipid designs that are thought to confer the property of fusibility to lipids include (i) unsaturation in the lipid tail, (ii) branching in the lipid carbon chain, (iii) polytailed lipids (e.g., 7C1 and G0-C14 lipids) (e.g., Dahlman et al., Nat Nanotechnol. 2014 Aug;9(8):648-655), and (iv) incorporation of polymeric lipids.

[0460] Protonable or cationic lipids containing unsaturated hydrocarbon chains are thought to provide LNPs with higher fluidity. Exemplary publications describing protonable and / or cationic lipids include U.S. Patent Publications 2006 / 0083780 and 2006 / 0240554, U.S. Patents 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613, and 5,785,992, and PCT Publication WO96 / 10390 (each of these disclosures is incorporated herein by reference in whole).

[0461] Exemplary protonable and / or cationic lipids comprise a protonable or cationic head group and optionally comprise one or more linear hydrocarbon chains of 10 to 20 carbon atoms, and optionally comprise one or more branched (optionally unsaturated) hydrocarbon chains of 12 to 30 carbon atoms, each of which is optionally interrupted by one or more functional groups selected from esters, ethers, amines (e.g., tertiary amines), amides, carbonates, carbamates, ureas, and disulfides. Protonable or cationic lipids may comprise a branched portion. In certain embodiments, the branched portion comprises a tertiary carbon atom, a quaternary carbon atom, a tertiary amine, a vicinal diol, an amide, a carbamate, or an acetal.

[0462] In certain embodiments, protonable or cationic lipids are protonable lipids containing amines ("protonable aminolipids"), e.g., tertiary amines. The protonation of amino groups at different pH levels is thought to provide further stabilization of load cargo (e.g., exogenous mRNA) in vivo (e.g., endosomes) while promoting cargo release within low pH compartments. In certain embodiments, protonable lipids have a pKa of protonable (e.g., amino) groups in the range of about 4 to about 7. Such lipids are primarily surface-neutralized at physiological pH, which is thought to reduce their sensitivity to clearance. The pKa measurement of lipids within lipid particles can be performed, for example, by using the method described in Cullis et al., (1986) Chem Phys Lipids 40, 127-144, using the fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS).

[0463] In certain embodiments, the protonable or cationic lipid is DLin-MC3-DMA, ALC-0315, SM-102, or LP-01: [ka] .

[0464] b. Polymer lipids In certain embodiments, LNPs containing polymer lipids are provided. The polymer lipids may be polymer-functionalized lipids in which polymers and lipids (e.g., hydrocarbon chains optionally interrupted by one or more intervening functional groups) are linked by covalent bonds with optionally intervening atoms. The polymer lipids optionally include branched portions. The lipids may be, for example, hydrocarbon chains optionally interrupted by one or more intervening functional groups. The polymer lipids generally contain uncharged hydrophilic moieties, such as PEG, GMI, or ATTA, which are thought to limit aggregation during the formulation of the LNP. Therefore, polymer lipids, when included in LNPs, are thought to reduce aggregation. In certain embodiments, the polymer lipid content in the LNP is selected to reduce particle aggregation. Examples of polymer lipids include polyethylene glycol (PEG)-modified lipids, monosialoganglioside GMIs, and polyamide oligomers ("PAOs"), as described in U.S. Patent No. 6,320,017. ATTA lipids are described, for example, in U.S. Patent No. 6,320,017, and PEG-functionalized lipids are described, for example, in U.S. Patents No. 5,820,873, 5,534,499, and 5,885,613. In certain embodiments, polymer lipids include one or more hydrocarbon chains interrupted by biodegradable functional groups (e.g., esters). Polymer lipids may include branched portions. In certain embodiments, branched portions include tertiary carbon atoms, quaternary carbon atoms, tertiary amines, vicinal diols, amides, carbamates, or acetals.

[0465] Lipids and optional branching regions are thought to influence the association strength between polymer lipids and LNPs. At least three features—lipid chain length, lipid chain stiffness (e.g., determined by saturation), and steric barrier head size (e.g., see U.S. Patent No. 5,820,873)—are thought to influence the exchange rate. In some therapeutic applications, it may be preferable for PEG-functionalized lipids to be rapidly lost from LNPs in vivo, and therefore PEG lipids will have relatively short lipid anchors. In other therapeutic applications, it may be preferable for nucleic acid-lipid particles to exhibit a longer plasma circulating lifetime, and therefore PEG lipids will have relatively long lipid anchors. mPEG(mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) is thought to remain associated with LNPs for several days in vivo. Other conjugates, such as PEG-CerC20, have similar retention capabilities. However, PEG-CerC14 is thought to be more rapidly exchanged from the formulation upon exposure to serum.

[0466] In certain embodiments, the polymer lipid is charge-neutral. In certain embodiments, the polymer lipid is amphoteric.

[0467] The polymer may be a hydrophilic polymer, such as poly(ethylene glycol) (also called poly(ethylene oxide) or PEG) ("PEG lipid"). In certain embodiments, PEG is a linear polymer of optionally substituted ethylene glycol or ethylene oxide. In certain embodiments, the PEG moiety is substituted with, for example, one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In certain embodiments, the PEG moiety includes a PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, for example, J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnic and biomedical applications (1992)). In certain embodiments, the PEG moiety is a PEG homopolymer.

[0468] Generally, the length of a PEG chain can be expressed by the number of repeating ethylene oxide units, or by the molecular weight of the Dalton units of the PEG portion of the polymer lipid. In a specific example, PEG-DMG 2000 has the following structure: [ka] In the formula, 2000 is the approximate molecular weight of the Dalton units of the PEG portion of the molecule. The length of the PEG chain may vary among individual lipids in the sample and can therefore be expressed as the average number of repeating units or the average molecular weight.

[0469] Generally, the length of a PEG chain can be expressed by the number of repeating ethyl oxide units, or by the molecular weight of the Dalton units of the PEG portion of the polymer lipid.

[0470] c. Charged neutral lipids and zwitterionic lipids In certain embodiments, LNPs include (charge) neutral or amphoteric lipids. Neutral or amphoteric lipids can generally be any lipid species that are uncharged or neutral amphoteric forms at physiological pH. Generally, neutral or amphoteric lipids include a polar head group and one or more (e.g., two) hydrophobic tail groups. Exemplary head groups include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and inositol.

[0471] In certain embodiments, the neutral lipid comprises two hydrocarbon groups, each optionally interrupted by a biodegradable moiety. Neutral or amphoteric lipids with diverse acyl chain groups of varying chain lengths and degrees of saturation are available or can be isolated or synthesized by known techniques. In certain embodiments, the neutral or amphoteric lipid comprises saturated fatty acids or mono- or di-unsaturated fatty acids. Furthermore, lipids having mixtures of saturated and unsaturated fatty acid chains can be used. In certain embodiments, the fatty acids are interrupted by biodegradable moieties such as esters.

[0472] In certain embodiments, neutral or amphoteric lipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), glycerophospholipids, sphingophospholipids, sphingolipids, phosphonolipids, natural lecithin, or hydrogenated phospholipids. Such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0473] The selection of neutral or amphoteric lipids is generally guided by considering, for example, LNP particle size and stability in circulation.

[0474] In certain embodiments, the neutral or amphoteric lipids are phospholipids. As used herein, “phospholipid” refers to a lipid comprising a hydrophilic phosphate head group and one or more hydrophobic tail groups. In some embodiments, phospholipids may facilitate fusion to membranes. For example, cationic phospholipids may interact with one or more loaded electrophospholipids of a membrane (e.g., a cell membrane or intracellular membrane). Fusion of phospholipids to a membrane may enable one or more components of an LNP (e.g., cargo) to be delivered across the membrane (e.g., to a cell).

[0475] In certain embodiments, the neutral or amphoteric lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0476] d. Cholesterol and its derivatives Cholesterol is a ubiquitous structural membrane lipid, and its role in lipid compositions is context-dependent. When combined with phospholipids having low gel-liquid crystal transition temperatures (Tm), cholesterol is thought to aid in the formation of a liquid-ordered phase, characterized by increased bilayer thickness and membrane stiffness. Cholesterol and other linear and / or branched hydrocarbon-containing (e.g., low-Tm) lipids are thought to bind in such a way that the cross-sectional area of ​​the lipids and cholesterol is less than the sum of their individual cross-sectional areas. However, when combined with high-Tm lipids, cholesterol is thought to enhance membrane fluidity and provide a narrower bilayer. In either case, cholesterol is thought to promote the liquid-ordered phase. Furthermore, cholesterol is thought to reduce the amount of surface-bound proteins and improve their circulating half-life. The amount of cholesterol in LNPs can be selected to match the cholesterol content of the membrane in vivo.

[0477] In some embodiments, LNPs containing cholesterol are provided.

[0478] In certain embodiments, the cholesterol derivatives include β-sitosterol, β-sitosterol acetate, β-sitosterol amino acid conjugate, fecosterol, ergosterol, 9,11-dehydroergosterol, campesterol, stigmasterol, brassicasterol, fucosterol, tomatidine, ursolic acid, daucosterol, cholesterol, 5-heptadecylresorcinol, cholesterol hemisuccinate, 6-keto-5α-hydroxycholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, and 7β,25-dihydroxycholesterol. Selected from terol, 27-hydroxycholesterol, 25-hydroxycholesterol, 20α-hydroxycholesterol, 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, 6-ketocholestanol, cholesteryl-(4'-hydroxy)-butyl ether, 5α-cholestane, cholestane, 5β-cholestanone, cholesteryl decanoate, vitamin D3, vitamin D2, calcipotriol, botulinum, luperol, ursolic acid, oleanolic acid, DC-cholesterol, BHEM-cholesterol, cholesteryl oleate, or combinations thereof. For example, see Paunovska, K. et al., Adv Mater. 2019 April; 31(14), Patel et al., Nat. Comm., (2020) 11: 983, Ni et al., Nat. Comm. 13, Article number: 4766 (2022), and Kim et al., ACS Nano 2022, 16(9), 14792-14806.

[0479] 2.LNP formation LNPs can be formed by any method known in the art, including but not limited to continuous mixing or direct dilution processes.

[0480] In certain embodiments, a method for preparing LNPs by a serial mixing method is provided. For example, the process includes providing an aqueous solution containing nucleic acids in a first reservoir, providing a lipid solution in a second reservoir, and mixing the organic lipid solution with the aqueous solution to rapidly generate LNPs that encapsulate cargoes. The lipid solution contains a lower alcohol such as ethanol. This process and apparatus for carrying out this process are described in detail in U.S. Patent Publication 2004 / 0142025 (the disclosure is incorporated herein by reference in its entirety). By mixing the aqueous solution containing cargoes with the organic lipid solution, the organic lipid solution undergoes serial dilution in the presence of a buffer (i.e., aqueous solution) to generate lipid nanoparticles.

[0481] In a particular embodiment, a method is provided for preparing LNPs by a direct dilution process, comprising forming an LNP solution and directly introducing the LNP solution into a recovery container containing a controlled volume of dilution buffer. The recovery container may include one or more elements configured to agitate the contents of the recovery container to facilitate dilution. In one embodiment, the amount of dilution buffer present in the recovery container is substantially equal to the volume of liposome solution introduced therein. As a non-limiting example, if a liposome solution in about 45% ethanol is introduced into a recovery container containing an equal volume of dilution buffer, smaller particles are advantageously obtained.

[0482] In a particular embodiment, a method is provided for preparing LNPs by a direct dilution process in which a third reservoir containing a dilution buffer is fluidically coupled to a second mixing region. In this embodiment, the LNP solution formed in the first mixing region is immediately and directly mixed with the dilution buffer in the second mixing region. In a preferred embodiment, the second mixing region includes a T-connector positioned so that the flows of the LNP solution and the dilution buffer meet as opposing 180° flows, although a connector providing a shallower angle can be used. In one embodiment, the flow rate of the dilution buffer supplied to the second mixing region is controlled to be substantially equal to the flow rate of the liposome solution introduced therefrom the first mixing region. Such control of the flow rate of the dilution buffer can advantageously allow for the formation of smaller particle sizes at reduced concentrations. A process and apparatus for carrying out the direct dilution process is described in U.S. Patent Publication 2007 / 0042031 (its disclosure is incorporated herein by reference in its entirety).

[0483] B. Viral vectors In certain embodiments, the editing system, guide nucleic acid binder, or nucleic acid encoding the guide may be introduced into a cell or organism via a vector. In certain embodiments, and in some cases, the vector may be a plasmid, minicircle, CELiD, virion derived from adeno-associated virus (AAV), or lentivirus.

[0484] Non-exclusive disclosure and incorporation by reference Each of the documents and patent publications listed herein is incorporated in whole by reference.

[0485] While certain compounds, compositions, and methods are specifically described herein according to certain embodiments, the following examples are merely illustrative of the compounds described herein and are not intended to limit them. Each of the references, GenBank accession numbers, ENSEMBL identifiers, etc., cited herein is incorporated herein by reference in whole.

[0486] The sequence listing accompanying this application identifies each nucleic acid sequence as either "RNA" or "DNA" as appropriate. However, those skilled in the art will readily understand that the designation of "RNA" or "DNA" to describe modified oligonucleotides is arbitrary in certain cases. For example, an oligonucleotide comprising a nucleoside containing a 2'-OH sugar moiety and a thymine base may be described as DNA having a modified sugar (i.e., a 2'-OH instead of one of the 2'-H in DNA) or as RNA having a modified base (i.e., thymine (5-methyluracil) instead of uracil in RNA). Certain nucleic acid compounds described herein comprise one or more nucleosides containing a modified sugar moiety having a 2'-substituent(s) that is neither OH nor H. Those skilled in the art will readily understand that labeling such nucleic acid compounds as "RNA" or "DNA" does not alter or limit the description of such nucleic acid compounds.

[0487] In this specification, descriptions of compounds having a nucleic acid sequence of a certain sequence number " refer only to the nucleic acid sequence. Therefore, unless otherwise specified, such descriptions of compounds by reference to a nucleic acid sequence of a certain sequence number do not limit the presence or absence of modifications to sugar or nucleoside bonds, or additional substituents such as conjugate groups. Furthermore, unless otherwise specified, the nucleic acid bases of compounds having a nucleic acid sequence of a certain sequence number " include such compounds having specific modified forms of nucleic acid bases as described herein.

[0488] In this specification, sugars, nucleoside bonds, and nucleic acid base modifications may be indicated within a nucleotide or nucleic acid base sequence, or in text accompanying the sequence (for example, in a table of compounds, or in separate text appearing above or below it).

[0489] While we have strived to accurately describe the compounds in the attached sequence listing, in the event of any discrepancy between the description in this specification and the description in the attached sequence listing, the description in the sequence listing shall prevail as the accurate description.

[0490] The compounds described herein include modifications in which one or more atoms are replaced with non-radioactive or radioactive isotopes of the indicated elements. For example, the compounds described herein, which include a hydrogen atom, 1 This includes all possible deuterium substitutions for each of the H hydrogen atoms. The isotopic substitutions included by the compounds herein are: 1 A substitute for H 2 H or 3 H, 12 A substitute for C 13 C or 14 C, 14 A substitute for N 15 N, 16 A substitute for O 17 O or 18 O, and, 32 A substitute 33 S, 34 S, 35 S, or 36 Examples include, but are not limited to, S. In certain embodiments, non-radioactive isotope substitution can confer novel properties to oligomeric compounds that are useful for use as therapeutic or research tools. In certain embodiments, radioactive isotope substitution can make compounds suitable for research or diagnostic purposes, such as imaging. [Examples]

[0491] Example 1: Computational prediction and characterization of Cas protein in HEK293 cells - expression and localization The metagenomics were assembled using publicly available datasets. CRISPR arrays and CRISPR cassettes containing nearby proteins were predicted and classified into subclasses and subtypes using CRISPRFinder and CRISPRone. For Class 2 type II CRISPR systems, protein domains were identified via Hidden Markov models, and sequences were aligned to Cas9 (SpCas9) of Streptococcus pyogenes. In addition, tracrRNAs were predicted using CRISPR arrays to predict antirepeats located at the 5' end of tracrRNAs and by searching for rho-independent terminations at the 3' end of tracrRNAs, characterized by GC-rich palindromic sequences followed by a series of U residues. Nineteen putative Cas proteins were identified. The corresponding Cas protein sequences and GeneArt (ThermoFisher) human codon-optimized nucleic acid sequences are provided in the sequence listing, as shown in the table below. The DNA sequences of the Cas proteins were cloned into the pTwistCMVpuro vector by Twist Biosciences.

[0492] To promote the nuclear localization of the enzymes, nucleoplasmin (SEQ ID NO: 3399) and c-Myc (SEQ ID NO: 3400) nuclear localization signals (NLS) were added to the N-terminus and C-terminus of each Cas protein, respectively. In addition, a HiBiT (Promega) tag was added to the C-terminus of the protein to detect its expression. These additional elements were linked to the protein via known protein linkers such as GGGS / (GS)3 / (GGGGS)3. The DNA sequences encoding the Cas proteins with NLS and HiBiT tags are provided in the sequence listing, as shown in DNA SEQ ID NOs 44-63 below. [Table 5]

[0493] Next, the expression and localization of each Cas protein were evaluated in HEK293 cells. 2 × 10 5HEK293 cells were transfected with 400 ng / well of codon-optimized DNA encoding the putative Cas protein using a 4D Nucleofector (Lonza), and the cells were seeded in 96-well plates post-transfection. Cells were harvested after either 48 or 72 hours. For each well, HiBit levels representing Cas protein expression were measured using a Nano-glo HiBit lysis detection system (Promega) according to the manufacturer's protocol. Cas protein expression was normalized to the number of cells measured by the CellTiter-Glo luminescence cell viability assay (Promega). In the table below, the expression of each Cas protein is expressed as the average of four measurements, as HiBit luminescence / CellTiter-Glo luminescence. A negative control "unprogrammed" group consisting of untransfected cells was included in the analysis. Compared to the unprogrammed negative control, HiBit expression was detected in all samples. Some samples showed high protein expression comparable to SpCas9, while others showed relatively low expression. [Table 6]

[0494] Next, the cellular localization of the Cas protein was examined by immunohistochemistry. HeLa cells were placed on 8-well chamber slides in a 1.5 × 10⁶ layer. 4Cells were seeded at a cell / well density and transfected with a codon-optimized Cas protein expression vector at 100 ng / well using 0.4 μL of BioT transfection reagent (Morganville Scientific). After 48 hours, cells were fixed in 4% formaldehyde and permeabilized with 0.1% Triton X-100 in PBS. After blocking the slides with 2% BSA in PBS at room temperature for 1 hour, the slides were incubated overnight at 4°C with mouse anti-HiBit antibody (Promega). The following day, the slides were washed and incubated for 1 hour with donkey anti-mouse secondary antibody AlexaFluor555 (1:1000) in combination with DAPI (1:1000). The slides were then washed and mounted in mounting medium. Images were acquired using EVOS cell imaging microscopy. The following table summarizes the cellular localization of Cas protein. The control SpCas9 group showed nuclear localization as expected. Eight Cas proteins showed nuclear localization, while the rest were mainly localized in the cytoplasm. [Table 7]

[0495] Example 2: Evaluation of Cas protein activity in HEK293T nuclear extract After confirming expression in HEK293T cells, the protein recognition region sequence was designed. For each Cas protein, the protein-specific crRNA and tracrRNA sequences were computationally predicted and linked to the "gaaa" tetraloop to obtain the putative guide protein recognition region shown in the table below. The "gaaa" tetraloop between the putative crRNA sequence and the tracrRNA sequence is represented in lowercase.

[0496] The above Cas proteins were screened for enzymatic activity using a cleavage assay in HEK293T cells. This assay measures the cleavage of synthetic tool DNA targets in nuclear extracts of HEK293T cells that express the Cas protein and have been transfected to transcribe its corresponding guide. The first round of screening (Figure 1) was performed using a system consisting of the Cas protein and a guide containing a general-purpose target recognition region bound to the protein recognition region. The second round of screening (Figure 2) was performed using a system consisting of the Cas protein and a guide containing a target recognition region optimized for the Cas protein, bound to the protein recognition region. [Table 8-1] [Table 8-2]

[0497] In preliminary experiments, each guide used a target recognition region known as a "general spacer sequence" (GSS), previously disclosed in Ran et al. Nature, 2015. The GSS target recognition regions are shown in the table below. In addition, other target recognition regions specific to each Cas protein were designed, as shown in the table below. The DNA sequences of each target recognition region are included in the sequence listing (SEQ ID NOs. 64-71). [Table 9]

[0498] Screening of Cas proteins using a general-purpose target recognition region Guides with a general-purpose target recognition region were designed by adding a general-purpose target recognition region sequence (sequence number 944) to the 5' end of a protein recognition sequence selected from the table above (sequence numbers 121-141).

[0499] A DNA template for in vitro synthesis of guide RNA was constructed. The target recognition sequence was added to the 5' end of the Cas protein recognition region to create the guide, and then the T7 promoter region (TAATACGACTCACTATA (SEQ ID NO: 72)) was added to the 5' end of the target recognition sequence. Furthermore, to improve binding to the T7 promoter, four nucleic acid bases AAGC were added to the 5' end of the T7 promoter region. The nucleic acid sequences of the DNA templates used for transcription of the guides listed in the table below are provided as SEQ ID NOs: 143-161, respectively. The HiScribe® T7 High Yield RNA Synthesis Kit (New England Bioloabs) was used for T7-based in vitro transcription of the templates. Each DNA template in the sequence listing represents the upper strand sequence, which has the same sequence as the RNA transcript at the end of transcription using the HiScribe® T7 High Yield RNA Synthesis Kit.

[0500] The guide sequences are shown in the table below. For each guide sequence in the table below, the target recognition region is shown in bold, underlined capital letters, the protein recognition region in regular capital letters, and the tetraloop in lowercase letters. The Cas protein for which each guide was designed is shown in the table below by its Cas protein ID and its corresponding sequence number. [Table 10-1] [Table 10-2]

[0501] The activity of each Cas protein was tested in the cleavage assay using the guides listed in the table above. The DNA target used in the cleavage assay was a 156 bp double-stranded DNA containing a PAM library followed by a general-purpose spacer (shown above). The target DNA sequence (5' to 3') is: [ka] In the sequence, underlined bold nucleic acid bases represent general-purpose spacers (target recognition regions), and 7N (N=A, C, G, T) represents the PAM library.

[0502] HEK293T cells were maintained in DMEM (10% FBS, 10 U / mL penicillin, 100 μg / mL streptomycin) and 1.5 × 10⁶ cells were measured. 6 Cells were seeded at a cell / plate density on 10 cm plates (Corning) and incubated for 24 hours. Cells were transfected with pTwistCMV-puro vector (5 μg / plate) expressing the Cas protein listed in Table 4 above using 20 μL of BioT transfection reagent (Morganville Scientific). HEK293T nuclear extracts were prepared using NE-PER nuclear and cytoplasmic extract reagent (Thermo Scientific) according to the manufacturer's protocol. Guide RNA was transcribed in vitro from the corresponding DNA template using the HiScribe T7 Quick high-yield RNA synthesis kit (New England Biolabs).

[0503] For each cleavage reaction, 5 μL of HEK293T nuclear extract expressing Cas protein and 200 nM in vitro transcription guide RNA were incubated in 3 μL of cleavage buffer NEBuffer r3.1 (New England BioLabs) at room temperature for 10 minutes to promote the formation of the Cas-guideribonucleoprotein (RNP) complex. Subsequently, 10 nM DNA template was added to the reaction mixture and incubated overnight at 37°C, followed by the addition of protease K and RNAse to clean up the reaction mixture. The DNA template was purified using DNA Clean & Concentrator-5 (Zymo Research). The digestion of the DNA template was analyzed and visualized using Agilent TapeStation. As shown in Figure 1, Cas proteins IONCAS009 and IONCAS016 were found to possess in vitro cleavage activity.

[0504] To further investigate the enzymatic activity of Cas proteins that showed relatively high expression but did not exhibit activity using a general-purpose spacer, additional screening was performed using enzyme-specific target recognition regions instead of the general-purpose spacer (target recognition region) sequence.

[0505] Screening of Cas proteins using Cas-specific target recognition regions The guide was designed by adding a Cas protein-specific target recognition region sequence (sequence numbers 945-951) selected from Table 8 above to the 5' end of a protein recognition sequence (sequence numbers 121-141) also selected from the table above.

[0506] A DNA template for in vitro synthesis of guide RNA was constructed. A guide was created by adding a Cas-specific target recognition sequence to the 5' end of the Cas protein recognition region described above, and then the T7 promoter region (TAATACGACTCACTATA (SEQ ID NO: 72)) was added to the 5' end of the target recognition sequence. In addition, since T7 strongly prefers to start with G and functions best with GG, GG was added to the 5' end of target recognition regions that start with nucleic acid bases other than G. For target recognition regions that start with G, one G was added to the 5' end. Furthermore, to improve binding to T7 polymerase, the four nucleic acid bases AAGC were added to the 5' end of the T7 promoter region. The nucleic acid sequences of the DNA templates used for transcription of the guides listed in the table below are provided as SEQ ID NOs: 162-174, respectively. The HiScribe® T7 High Yield RNA Synthesis Kit (New England Bioloabs) was used for T7-based in vitro transcription of the templates. Each DNA template in the sequence listing represents an upper strand sequence and has the same sequence as the RNA transcript at the end of transcription using the HiScribe® T7 high-yield RNA synthesis kit.

[0507] The guide sequences are shown in the table below. For each guide sequence in the table below, the target recognition region is shown in bold, underlined capital letters, the protein recognition region in regular capital letters, and the tetraloop in lowercase letters. The Cas protein for which each guide was designed is shown in the table below by its Cas protein ID and sequence number. [Table 11-1] [Table 11-2]

[0508] The cleavage assay was repeated as described above with the following modifications: the guide used consisted of the Cas-specific guides listed in the table above, and the DNA target used consisted of a Cas-specific target recognition region attached to a 7nt degenerate PAM sequence. When the Cas-specific target recognition region was used, the Cas proteins IONCAS008 and IONCAS013 showed in vitro cleavage activity in nuclear extracts (Figure 2).

[0509] Example 3: Identification of the PAM sequence of the Cas protein The PAM sequences recognized by each Cas protein exhibiting in vitro enzymatic activity were determined by a dropout assay (Ran, FA et al. Nature. 2015, 520, 186-191). In the dropout assay, the uncut fraction of the DNA template was amplified by PCR using Integrated DNA Technologies (IDT) i5 / i7 index primers. The amplicons were sequenced via Illumina MiSeq. Given a single-ended MiSeq sequencing run, the data were demultiplexed using either the MiSeq machine or Illumina bcl2fastq conversion software (https: / / support.illumina.com / sequencing / sequencing_software / bcl2fastq-conversion-software / downloads.html) to generate a FASTQ file. A custom program was created to extract the 7-mer PAM sequence located 3' to the known spacer sequence from the FASTQ file. For each extracted PAM sequence in the sample, the counts per million (CPM) value was calculated by dividing the number of reads containing the PAM sequence by the total number of reads in the sample. The relative PAM sequence depletion frequency was calculated by dividing the CPM value in the Cas protein-treated sample by the corresponding CPM value in the untreated control sample. PAM sequence motifs with different depletion frequency thresholds were then generated using the Python Logomaker software library (Tareen and Kinney 2019), available at https: / / logomaker.readthedocs.io / en / latest / . The consensus PAM sequences for the Cas proteins IONCAS008, IONCAS009, and IONCAS016 are summarized in the table below and shown in Figure 3. [Table 12]

[0510] Example 4: Evaluation of intracellular activity of Cas protein against gene targets in HEK293 cells The enzymatic activity of the above Cas proteins was evaluated in HEK293 cells, and editing in six genomic regions was measured. For each Cas protein, a guide was designed containing a target recognition region sequence designed for a genomic region ligated to the 5' end of a protein recognition region sequence selected from the above examples. The guides were then evaluated in HEK293 cells using an in vitro cleavage assay.

[0511] Six genomic regions widely used in SpCas9 research were selected as targets for evaluating intracellular Cas protein activity. These regions are located within the human genomic loci of AAVS1, EMX1, FANCF, HBB, HEKSite4, and HPRT. For each putative Cas protein and guide system, target recognition regions were designed using CHOPCHOP v3 (Labun et al. Nucleic Acids Res, 2019) and Benchling software (shown in the table below). The Cas proteins and gene targets for which each target recognition region was designed are shown in the table below. The DNA sequences of each target recognition region are included in the sequence listing (SEQ ID NOs: 175-199). [Table 13]

[0512] Guides were designed by adding the Cas protein-specific target recognition region sequence from the table above to the 5' end of a protein recognition sequence (SEQ ID NOs. 121-141) selected from the table above. The guide sequences are shown in the table below. For each guide sequence, the target recognition region is shown in bold, underlined capital letters, the protein recognition region in regular capital letters, and the tetraloop in lowercase letters. The Cas protein for which each guide was designed is shown in the table below by its Cas protein ID and SEQ ID NO. The gene target for which each guide was designed is also shown in the table below. The DNA sequence for each guide is included in the sequence listing (SEQ ID NOs. 200-229). [Table 14-1] [Table 14-2] [Table 14-3]

[0513] Plasmids expressing the guide were designed by cloning the guide sequence into a U6 promoter-driven vector for transcription by RNA polymerase III, and ordered from Twist BioScience. The U6 promoter sequence (GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCg; SEQ ID NO: 75) was added to the 5' end of the guide sequence. Here, for guides with 5'-GG, the last lowercase 'g' was removed. The transcription termination sequence (GTCTAGAGGTAC; SEQ ID NO: 76) was added to the 3' end.

[0514] 2 x 10 5 Nine HEK293 cells were transfected using a 4D Nucleofector (Lonza) with 400 ng / well of codon-optimized DNA encoding the Cas protein and 400 ng / well of a guide expression plasmid. The cells were seeded in 96-well plates after transfection. Analysis included two negative controls: cells transfected with SpCas9 / scrambled spgRNA, and untransfected cells. After a 48 or 72-hour incubation period, genomic DNA was extracted from the cells using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's recommended protocol. Genomic PCR was performed using primers spanning the target region, and libraries were constructed using IDT's i5 / i7 index primers. Amplicons were sequenced via MiSeq (Illumina). For a given paired-end MiSeq sequencing run, the data were demultiplexed as described above to generate a FASTQ file. All sample sequencing data was processed and analyzed using the CRISPRessoBatch module of the CRISPResso2 software (Clement et al. Nat Biotechnol, 2019). A custom script was created to calculate the editing percentage based solely on InDel from the CRISPResso2 analysis results. The gene editing quantification window was set to approximately 40 nt in length, with approximately 10 nt upstream and 10 nt downstream of a guide RNA sequence of approximately 20 nt. To visualize the alignment of low-frequency InDel reads, the minimum threshold for reported reads was set to 0.01% (default is 0.20%), and the maximum number of reported alignments was set to 100 (default is 50).

[0515] The results are summarized in the table below as the percentage of InDel (%). "ND" indicates that data was not available. [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20]

[0516] Example 5: Computational prediction of Cas protein and associated guides The metagenomics were assembled using publicly available datasets. CRISPR arrays and CRISPR cassettes containing nearby proteins were predicted and classified into subclasses and subtypes using CRISPRFinder and CRISPRone. For Class 2 type II CRISPR systems, protein domains were identified via hidden Markov models and sequences were aligned to Streptococcus pyogenes Cas9 (SpCas9). In addition, tracrRNAs were predicted by using CRISPR arrays to predict antirepeats located at the 5' end of tracrRNAs and by searching for rho-independent terminations at the 3' end of tracrRNAs, characterized by GC-rich palindromic sequences followed by a series of U residues.

[0517] Following these analyses, 93 putative Cas proteins were identified. The corresponding Cas protein sequences and GeneArt (ThermoFisher) human codon-optimized nucleic acid sequences are provided in the sequence listing, as shown in the table below. The DNA sequences of the Cas proteins were cloned into the pTwistCMVpuro vector by Twist Biosciences.

[0518] To promote the nuclear localization of the enzymes, nucleoplasmin (SEQ ID NO: 3399) and c-Myc (SEQ ID NO: 3400)NLS were added to the N-terminus and C-terminus of each Cas protein, respectively. In addition, a HiBiT (Promega) tag was added to the C-terminus of the protein to detect expression. These additional elements were linked to the protein via known protein linkers such as GGGS / (GS)3 / (GGGGS)3. The DNA sequences encoding the Cas proteins with NLS and HiBiT tags are provided in the sequence listing, as shown in DNA SEQ ID NOs 231-509 below. [Table 21-1] [Table 21-2] [Table 21-3]

[0519] Example 6: Evaluation of Cas protein activity in HEK293T nuclear extract The protein recognition region sequences of the guide were designed. For each Cas protein, the sequences of protein-specific crRNA and tracrRNA were computationally predicted and linked to the "gaaa" tetraloop to obtain the putative guide protein recognition elements shown in the table below. The "gaaa" tetraloop between the putative crRNA sequence and the tracrRNA sequence is represented by lowercase nucleotides.

[0520] The above Cas proteins were screened for enzymatic activity using the HEK293T cell cleavage assay described herein. This assay measures the cleavage of synthetic tool DNA targets in nuclear extracts of HEK293T cells that express Cas proteins and have been transfected to transcribe their corresponding guides. The guides for each Cas protein, including the target recognition region ligated to the 5' end of the protein recognition region, were designed as described below. [Table 22-1] [Table 22-2] [Table 22-3] [Table 22-4] [Table 22-5] [Table 22-6]

[0521] Screening of Cas proteins using a general-purpose target recognition region Guides with a general-purpose target recognition region were designed by adding a general-purpose target recognition region sequence (sequence number 944) to the 5' end of a protein recognition region sequence selected from the table above (sequence numbers 510-602).

[0522] DNA templates for in vitro synthesis of guide RNA were constructed. A guide was created by adding the target recognition sequence to the 5' end of the Cas protein recognition region, and then the T7 promoter region (TAATACGACTCACTATA (SEQ ID NO: 72)) was added to the 5' end of the target recognition sequence. Furthermore, to improve binding to the T7 promoter, four nucleotides, AAGC, were added to the 5' end of the T7 promoter region. The nucleic acid sequences of the DNA templates used for transcription of guides C20g to C114g, listed in the table below, are provided as SEQ ID NOs. 603 to 695, respectively. The HiScribe® T7 High Yield RNA Synthesis Kit (New England Biolabs) was used for T7-based in vitro transcription of the templates. Each DNA template in the sequence listing represents the upper strand sequence, which has the same sequence as the RNA transcript at the end of transcription using the HiScribe® T7 High Yield RNA Synthesis Kit.

[0523] The guide sequences are shown in the table below. For each guide sequence in the table below, the target recognition region is shown in bold, underlined capital letters, the protein recognition region in regular capital letters, and the tetraloop in lowercase letters. The Cas protein for which each guide was designed is shown in the table below by its Cas protein ID and its corresponding sequence number. [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4] [Table 23-5] [Table 23-6] [Table 23-7]

[0524] The above Cas proteins were screened for enzymatic activity using an in vitro cleavage assay, and the synthesized DNA targets were incubated with nuclear extracts of HEK293T cells transfected with the pTwist-CMV-puro-Cas protein, and their corresponding in vitro transcription guide RNAs. The DNA targets were 156 bp double-stranded DNA containing a PAM library followed by a general-purpose spacer. The target DNA sequence is as described above (SEQ ID NO: 230).

[0525] HEK293T cells were maintained in DMEM (10% FBS, 10 U / mL penicillin, 100 μg / mL streptomycin) and 1.5 × 10⁶ cells were measured. 6Cells were seeded at a cell / plate density in 10 cm plates (Corning) and incubated for 24 hours. Cells were transfected with pTwistCMV-puro vector (5 μg / plate) expressing the Cas proteins listed in Table 2 above using 20 μL of BioT transfection reagent (Morganville Scientific). HEK293T nuclear extracts were prepared using NE-PER nuclear and cytoplasmic extract reagent (Thermo Scientific) according to the manufacturer's protocol. Guide RNA was transcribed in vitro from the corresponding DNA templates using the HiScribe T7 Quick high-yield RNA synthesis kit (New England Biolabs).

[0526] For each cleavage reaction, 5 μL of HEK293T nuclear extract expressing Cas protein and 200 nM in vitro transcription guide RNA were incubated in 3 μL of cleavage buffer NEBuffer r3.1 (New England BioLabs) at room temperature for 10 minutes to promote the formation of the Cas-guide ribonucleoprotein (RNP) complex. Subsequently, 10 nM DNA template was added to the reaction mixture and incubated overnight at 37°C, followed by the addition of protease K and RNAse to clean up the reaction mixture. The DNA template was purified using DNA Clean & Concentrator-5 (Zymo Research). The digestion of the DNA template was analyzed and visualized using Agilent TapeStation.

[0527] As shown in Figures 4a-4c, the Cas proteins IONCAS030, IONCAS031, IONCAS034, IONCAS036, IONCAS050, IONCAS056, IONCAS057, IONCAS058, IONCAS061, IONCAS064, IONCAS065, IONCAS069, IONCAS073, IONCAS074, IONCAS075, IONCAS079, IONCAS080, IONCAS083, IONCAS088, IONCAS090, IONCAS091, IONCAS092, IONCAS093, IONCAS105, and IONCAS114 were found to possess in vitro cleavage activity.

[0528] Example 7: Identification of the PAM sequence of the Cas protein The PAM sequence of the Cas protein exhibiting in vitro enzymatic activity was determined by a dropout assay (Ran, FA et al. Nature. 2015, 520, 186-191). In the dropout assay, the uncut fraction of the DNA template was amplified by PCR using Integrated DNA Technologies (IDT) i5 / i7 index primers. The amplicon was sequenced via Illumina MiSeq. Given a single-ended MiSeq sequencing run, the data was demultiplexed using either the MiSeq machine or Illumina bcl2fastq conversion software (https: / / support.illumina.com / sequencing / sequencing_software / bcl2fastq-conversion-software / downloads.html) to generate a FASTQ file. A custom program was created to extract the 7-mer PAM sequence located 3' to the known spacer sequence from the FASTQ file. For each extracted PAM sequence in the sample, the counts per million (CPM) value was calculated by dividing the number of reads containing the PAM sequence by the total number of reads in the sample. The relative PAM sequence depletion frequency was calculated by dividing the CPM value in the Cas protein-treated sample by the corresponding CPM value in the untreated control sample. Then, PAM sequence motifs with different depletion frequency thresholds were generated using the Python Logomaker software library (Tareen and Kinney 2019), available at https: / / logomaker.readthedocs.io / en / latest / .The consensus PAM sequences for the Cas proteins IONCAS030, IONCAS031, IONCAS034, IONCAS036, IONCAS050, IONCAS056, IONCAS057, IONCAS058, IONCAS061, IONCAS064, IONCAS065, IONCAS069, IONCAS073, IONCAS074, IONCAS075, IONCAS079, IONCAS080, IONCAS083, IONCAS088, IONCAS090, IONCAS091, IONCAS092, IONCAS093, IONCAS105, and IONCAS114 are summarized in the table below. [Table 24]

[0529] Example 8: Guide design and efficacy in HEK293 cells A guide containing a target recognition region sequence ligated to the 5' end of a protein recognition region sequence was designed for Cas proteins selected from the above examples. The guide was optimized by adjusting various parameters, including the length of the target recognition region, the length of the first stem-loop in the protein recognition region, the substitution of AU or UA base pairs with CG base pairs in the predicted stem, the substitution of loops and linkers with different sequences, and the overall length of the protein recognition region. The guide was then evaluated in HEK293 cells using an in vitro cleavage assay.

[0530] Plasmids expressing the guide were designed by cloning the guide sequence into a U6 promoter-driven vector for RNA polymerase III transcription and ordered from Twist BioScience. The U6 promoter sequence (SEQ ID NO: 75) was added to the 5' end of the guide sequence, and for guides that already had a 5'-GG, the final 3'-G was removed. The transcription termination sequence (SEQ ID NO: 76) was added to the 3' end.

[0531] 2 x 10 5HEK293 cells were transfected using a 4D Nucleofector (Lonza) with 400 ng / well of codon-optimized DNA encoding the Cas protein (shown in the table below) and 400 ng / well of guide expression plasmid (shown in the table below). The cells were seeded in 96-well plates after transfection. After a 48 or 72-hour incubation period, genomic DNA was extracted from the cells using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's recommended protocol. For the targets shown in the table below, genomic PCR was performed using primers spanning the target region, and libraries were constructed using IDT's i5 / i7 index primers. Amplicons were sequenced via MiSeq (Illumina). For a given paired-end MiSeq sequencing run, the data were demultiplexed as described above to generate a FASTQ file. All sample sequencing data was processed and analyzed using the CRISPRessoBatch module of the CRISPResso2 software (Clement et al. Nat Biotechnol, 2019), and InDel analysis was performed as described above.

[0532] Optimization of the length of the target recognition region Target recognition region sequences with a length of 20–24 nt were constructed using the PAM sequences determined for each Cas protein and target pair. These target recognition region sequences can be found in sequence listings, as shown in the table below (SEQ ID NOs: 696–887). The guide was designed by appending a 20–24 nt target recognition region sequence to the 5' end of a protein recognition sequence selected from the above examples.

[0533] The guides listed in the table below were constructed from the target recognition region identified by the ID and sequence number in the "Target Recognition Region" column and the protein recognition region identified by the ID and sequence number in the "Protein Recognition Region" column. In the table below, the target for which each guide was designed is shown in the column labeled "Target," and the length of the target recognition region is shown in the column labeled "Length." The complete sequences of the guides can be found in the sequence listings, as shown in the column labeled "Guide."

[0534] As described above, Cas proteins and guides were tested for in vitro activity in HEK293 cells, and the results are shown in the table below. For each Cas protein and guide system, the relative amount of InDel was calculated by dividing the %InDel of the target of a given Cas protein and guide system by the %InDel of that target of the Cas protein and guide system when the length of the target recognition region is 20 nt. The results are shown in the table below as "Relative Indel relative to 20 nt". "ND" indicates that data was not available. [Table 25] [Table 26] [Table 27] [Table 28] [Table 29] [Table 30] [Table 31] [Table 32] [Table 33] [Table 34] [Table 35] [Table 36] [Table 37]

[0535] Optimization of the length of the protein recognition region for the first stem-loop. The protein recognition region sequences were modified by altering the repeat:anti-repeat (R:AR) length by varying the length of the upper stem loop at the 5' end of the protein recognition region, as predicted by ViennaFold software. The R:AR length is the number of nucleotides in the protein recognition region at the 5' end of the "gaaa" tetraloop, and is shown in the column labeled "R:AR Length" in the table below. The complete protein recognition region sequences associated with each R:AR modification can be found in the sequence listing (SEQ ID NOs. 961-1019), as shown in the table below. Guides were then designed by appending target recognition region sequences selected from the above examples to the 5' end of protein recognition sequences with various R:AR lengths.

[0536] The guides listed in the table below were constructed from the target recognition region identified by the ID and sequence number in the "Target Recognition Region" column and the protein recognition region identified by the ID and sequence number in the "Protein Recognition Region" column. In the table below, the target for which each guide was designed is shown in the column labeled "Target," and the R:AR length of the protein recognition region is shown in the column labeled "R:AR Length." The complete sequences of the guides can be found in the sequence listings (sequence numbers 1601-1766), as shown in the column labeled "Guide."

[0537] As described above, Cas proteins and guides were tested for in vitro activity in HEK293 cells, and the results are shown in the table below. For each Cas protein and guide system, the relative InDel was calculated by dividing the %InDel of the target of a given Cas protein and guide system by the %InDel of that target of the Cas protein and guide system with its original R:AR length (15 nt for IONCAS009, 16 nt for other Cas proteins). The results are shown as "Relative InDel" in the table below. [Table 38] [Table 39] [Table 40] [Table 41] [Table 42] [Table 43] [Table 44] [Table 45] [Table 46] [Table 47] [Table 48] [Table 49] [Table 50]

[0538] Optimization of the protein recognition region: The second stem-loop Further modifications to the protein recognition region were explored by modifying the sequences of the first linker (L1) and the second stem-loop (S2 and S2', as well as H2) of the protein recognition region of C9gRNA (SEQ ID NO: 131, as described herein). In the table below, the column labeled "Modification" shows the specific modifications. The complete protein recognition region sequences associated with each SL-1 modification can be found in the sequence listings (SEQ ID NOs: 1016-1019), as shown in the table below. Guides were then designed by appending a target recognition region sequence selected from the above examples to the 5' end of the modified protein recognition sequence.

[0539] The guides listed in the table below were constructed from the target recognition region identified by the ID and sequence number in the "Target Recognition Region" column and the protein recognition region identified by the ID and sequence number in the "Protein Recognition Region" column. In the table below, the target for which each guide was designed is shown in the column labeled "Target," and modifications in the protein recognition region are shown in the column labeled "Modification." The complete sequences of the guides can be found in the sequence listings (sequence numbers 1767-1778), as shown in the column labeled "Guide."

[0540] As described above, Cas proteins and guides were tested for in vitro activity in HEK293 cells. The percentage of insertions and deletions (%InDel) for each target for each IONAS009 protein and guide system is reported in the table below. [Table 51]

[0541] Optimization of the length of the protein recognition region The protein recognition region sequence was modified by cutting the 3' end stem loop in the guide or by removing a base pair in the 3' end stem loop to effectively shorten the overall sequence length. The full length of the protein recognition region sequence is shown in the column labeled "Length" in the table below. The complete protein recognition region sequences associated with each modification can be found in the sequence listing (SEQ ID NOs. 1020–1051) as shown in the table below.

[0542] An additional 20nt-length target recognition region sequence was constructed using the PAM sequence determined for each Cas protein and target pair. The target recognition region sequences can be found in the sequence listing, as shown in the table below (SEQ ID NOs: 888–890). Guides were then designed by appending the target recognition region sequences to the 5' end of protein recognition sequences of varying lengths.

[0543] The guides listed in the table below were constructed from the target recognition region identified by the ID and sequence number in the "Target Recognition Region" column and the protein recognition region identified by the ID and sequence number in the "Protein Recognition Region" column. In the table below, the target for which each guide was designed is shown in the column labeled "Target," and the length of the protein recognition region sequence is shown in the column labeled "Length." The complete sequences of the guides can be found in the sequence listings (sequence numbers 1409, 1413, 1417, 1856, 1860, 1868, 1872, 1889-1949), as shown in the column labeled "Guide."

[0544] As described above, Cas proteins and guides were tested for in vitro activity in HEK293 cells. The percentage of insertions and deletions (%InDel) for each target for each Cas protein and guide system is reported in the table below. [Table 52] [Table 53] [Table 54] [Table 55] [Table 56] [Table 57] [Table 58] [Table 59] [Table 60]

[0545] Optimization of the protein recognition region sequence The protein recognition region sequence was modified (using ViennaFold) by swapping various base pairs in the predicted stem-loop stem, for example, AU to GC, or GU fluctuation pairs to GC. The complete protein recognition region sequence can be found in the sequence listing (SEQ ID NOs. 1053–1312), as shown in the table below. The guide was then designed by appending the target recognition region sequence to the 5' end of the protein recognition sequence.

[0546] The guides listed in the table below were constructed from the target recognition regions identified by the ID and sequence number in the "Target Recognition Region" column and the protein recognition regions identified by the ID and sequence number in the "Protein Recognition Region" column. In the table below, the target for which each guide was designed is shown in the column labeled "Target". The complete sequences of the guides can be found in the sequence listings (sequence numbers 1950-2471), as shown in the column labeled "Guide".

[0547] As described above, Cas proteins and guides were tested for in vitro activity in HEK293 cells. For each Cas protein and guide system, the percentage of insertions and deletions (%InDel) relative to each target is reported in the table below. The relative amount of InDel was also calculated by dividing the %InDel of the target of a given Cas protein and guide system by the %InDel of that target of the Cas protein and guide system with the original protein recognition region. The results are shown in the table below as "Relative InDel". [Table 61] [Table 62-1] [Table 62-2] [Table 63-1] [Table 63-2] [Table 64-1] [Table 64-2] [Table 65-1] [Table 65-2] [Table 66-1] [Table 66-2] [Table 67] [Table 68-1] [Table 68-2] [Table 69-1] [Table 69-2] [Table 70-1] [Table 70-2] [Table 71-1] [Table 71-2]

[0548] Example 9: Design and efficacy of modification guides in HepG2 cells, and LNP delivery Modification guides containing a target recognition region sequence ligated to the 5' end of the protein recognition region sequence were designed for Cas proteins selected from the above examples and then purchased from IDT. The guides were optimized by adjusting the length of the target recognition region, the length of the protein recognition region, and the sequence of the protein recognition region. The guides were then screened for enzyme activity in an in vitro assay, and HepG2 cells were incubated with the Cas protein and the LNP formulation of the guide system.

[0549] The guides were designed by their sequence numbers according to the sequences shown in the table below and ordered from IDT. Each guide in the table below has the following modifications: the 1st and 2nd nucleosides at the 5' end are 2'-OMe sugar moieties, with each nucleoside linked to the next nucleoside by a phosphorothioate nucleoside linkage; the last two nucleosides at the 3' end are 2'-OMe sugar moieties, with each nucleoside linked to the previous nucleoside by a phosphorothioate nucleoside linkage.

[0550] Human HepG2 cells were passaged in 96-well plates at a density of 20,000 cells per well and cultured in DMEM medium containing 10% FBS and 1% PenStrep. After 24 hours, the medium was changed. RNA / LNP complexes were generated by mixing RNA (Cas protein mRNA / guide in a 1:2 ratio) with an LNP preparation (Kazemian, et al., Mol. Pharmaceuticals, 2022, 19, 1669-1689; Albertsen et al., Adv. Drug Deliv. Rev. 2022, 118, 114416; Cullis et al., Nat. Rev. Drug Discov. 2024, 23, 709-722). After incubation for 15 minutes, the mixture was added to the medium at the doses shown in the table below. Cells were harvested after 72 hours for InDel analysis and performed as described above herein.

[0551] Optimization of the length of the target recognition region Target recognition region sequences with a length of 20–25 nt were selected from the examples above or designed using the PAM sequences determined for each Cas protein and target pair. The target recognition region sequences can be found in the sequence listings, as shown in the table below.

[0552] The protein recognition region sequences for each Cas protein were designed, and the UUUUUU(6U) sequence was cleaved at the 3' end. The protein recognition region sequences can be found in the sequence listings, as shown in the table below.

[0553] The guide was designed by adding a target recognition region sequence with a length of 20–25 nt to the 5' end of the protein recognition sequence, with or without the presence of a 6U sequence at the 3' end.

[0554] The guides listed in the table below were constructed from the target recognition regions identified by the ID and sequence number in the "Target Recognition Region" column and the protein recognition regions identified by the ID and sequence number in the "Protein Recognition Region" column. In the table below, the length of the target recognition region sequence is shown in the column labeled "Length," and the presence of 6U at the 3' end of the protein recognition sequence is shown in the column labeled "3' End." Fully annotated sequences describing the nucleic acid sequences of the guides and modifications on the guides can be found in the sequence listings, as shown in the guide columns labeled "Sequence Number" and "Modified Sequence Number," respectively.

[0555] As described above, Cas proteins and guides were tested for in vitro activity in HepG2 cells. The percentage of insertions and deletions (%InDel) for each target for each Cas protein and guide system is reported in the table below. [Table 72] [Table 73] [Table 74] [Table 75] [Table 76] [Table 77] [Table 78] [Table 79] [Table 80] [Table 81] [Table 82] [Table 83] [Table 84] [Table 85]

[0556] Optimization of the protein recognition region sequence The protein recognition region sequences were modified by swapping various base pairs in the stem-loop, for example, from AU to CG or from GU to GC. The selected protein recognition sequences are shown in the table below. As shown in the table below, all protein recognition region sequences can be found in the sequence listings. Next, guides were designed by appending the target recognition region sequence to the 5' end of the protein recognition sequence. In the table below, the "gaaa" tetraloop is represented in lowercase. [Table 86]

[0557] The guides listed in the table below were constructed from target recognition regions identified by the ID and sequence number in the "Target Recognition Region" column and protein recognition regions identified by the ID and sequence number in the "Protein Recognition Region" column. In the table below, the target for which each guide was designed is shown in the column labeled "Target". Fully annotated sequences describing the nucleic acid sequences of the guides and modifications on the guides can be found in sequence listings, as shown in the guide columns labeled "Sequence Number" and "Modified Sequence Number," respectively.

[0558] As described above, Cas proteins and guides were tested for in vitro activity in HepG2 cells. The percentage of insertions and deletions (%InDel) for each target for each Cas protein and guide system is reported in the table below. [Table 87] [Table 88] [Table 89-1] [Table 89-2] [Table 90-1] [Table 90-2] [Table 91-1] [Table 91-2] [Table 92] [Table 93-1] [Table 93-2]

[0559] Example 10: Design and efficacy of Cas fusion protein and modification guide for epigenetic repression of PCSK9 in Hepa1-6 cells, and LNP delivery The above Cas protein was inserted into a Cas fusion protein containing a catalytically inactive Cas protein and a DNA demethylation domain. A modification guide containing a target recognition region sequence that targets PCSK9, bound to the 5' end of the protein recognition region sequence, was designed for the Cas fusion protein and purchased from IDT. The Cas fusion protein and modification guide system were tested for epigenetic repression of PCSK9 in mouse Hepa1-6 cells.

[0560] Design of Cas fusion proteins for DNA methylation The nuclease-dead versions of the Cas proteins described herein were designed by replacing the aspartic acid and histidine residues involved in nuclease activity with alanine. For the Cas protein IONCAS009, the point mutations are D11A and H871A. For the Cas protein IONCAS075, the point mutations are D11A and H868A. To confer DNA methylation activity, the C-terminal domain of Dnmtl3l (3A3L, SEQ ID NO: 958) was fused to the N-terminus of the catalytically inactive Cas protein via the XTEN80 linker sequence (SEQ ID NO: 959) to add flexibility and spacing. The 3A3L domain and XTEN80 linker have been previously disclosed in International Patent Application No. WO2023 / 215711.

[0561] The sequences of the Cas fusion proteins are provided in the sequence listing, as shown in the table below. The DNA sequences of the Cas fusion proteins were cloned into the VectorBuilder pmRVac vector for in vitro transcription (SEQ ID NO: 77). To promote the nuclear localization of the enzymes, nucleoplasmin (SEQ ID NO: 3399) and c-Myc (SEQ ID NO: 3400)NLS were added to the N-terminus and C-terminus of each Cas protein sequence, respectively. In addition, a HiBiT (Promega) tag was added to the C-terminus of the protein to detect expression. These additional elements were linked to the proteins via known protein linkers such as GGGS / (GS)3 / (GGGGS)3. A poly-A tail was added to the 3' end of the DNA sequences encoding the Cas proteins with the NLS and HiBiT tags. These DNA sequences are provided in the sequence listing, as shown in SEQ ID NOs: 956-957 below. [Table 94]

[0562] Design and efficacy of Cas fusion proteins and guide systems targeting PCSK9 The target recognition region sequence for PCSK9 was designed using the PAM sequences determined for each Cas fusion protein and PCSK9. The target recognition region sequences can be found in the sequence listings, as shown in the table below. Next, guides were designed by appending the target recognition region sequence to the 5' end of the protein recognition sequence C9g-NU (SEQ ID NO: 1052) for IONCAS009-d or the protein recognition sequence C75g-46 (SEQ ID NO: 1375) for IONCAS075-d. A guide for the positive control SpCas9 was designed similarly.

[0563] Each guide in the table below has the following modifications: the 1st and 2nd nucleosides at the 5' end are 2'-OMe sugar moieties, with each nucleoside linked to the next nucleoside via a phosphorothioate nucleoside linkage; and the last two nucleosides at the 3' end are 2'-OMe sugar moieties, with each nucleoside linked to the previous nucleoside via a phosphorothioate nucleoside linkage.

[0564] The guides listed in the table below were constructed from the target recognition regions identified by the ID and sequence number in the "Target Recognition Region" column and the protein recognition regions identified by the ID and sequence number in the "Protein Recognition Region" column. Fully annotated sequences describing the nucleic acid sequences of the guides and modifications on the guides can be found in the sequence listings, as shown in the guide columns labeled "Sequence Number" and "Modified Sequence Number," respectively.

[0565] Mouse Hepa1-6 cells were seeded in 96-well plates at a density of 20,000 cells per well 2–4 hours prior to transfection. RNA / LNP complexes were generated by mixing 220 ng of Cas fusion protein mRNA and 440 ng of guide RNA with the LNP preparation. After incubation at room temperature for 10 minutes, the mixture was added to the culture medium.

[0566] Samples were collected 3 days after transfection. RNA was extracted using RLT buffer (Thermo Fisher) and a Pall AcroPrep Advance 96-well plate. PCSK9 RNA was quantified by quantitative PCR using the TaqMan primer probe set Mm00463746_m1 (Thermo Fisher). PCSK9 RNA levels were normalized to GAPDH RNA using the TaqMan primer probe set Mm99999915_g1 (Thermo Fisher) or to ACTB RNA using the TaqMan primer probe set Mm02619580_g1 (Thermo Fisher). PCSK9 RNA expression is shown in the table below as the percentage of PCSK9 RNA (%UTC) relative to the amount of PCSK9 RNA in untreated control cells. [Table 95] [Table 96]

[0567] Duration of action of Cas fusion proteins and guide systems targeting PCSK9 Guides were selected from the above activity screen and their activity over time in mouse Hepa1-6 cells was tested. Mouse Hepa1-6 cells were seeded in 96-well plates at a density of 20,000 cells per well 2–4 hours before transfection. RNA / LNP complexes were generated by mixing 220 ng of Cas fusion protein mRNA and 440 ng of guide RNA with the LNP preparation. SpCas9 and guide SpPCSKg were included as controls. After incubation at room temperature for 10 minutes, the mixtures were added to the culture medium.

[0568] Samples were collected at the time points shown in the table below. Cells were passaged every 2-3 days to maintain optimal cell density. RNA was extracted using RLT buffer (Thermo Fisher) and Pall AcroPrep Advance 96-well plates. PCSK9 RNA was quantified by quantitative PCR using the TaqMan primer probe set Mm00463746_m1 (Thermo Fisher). PCSK9 RNA levels were normalized to GAPDH RNA using the TaqMan primer probe set Mm99999915_g1 (Thermo Fisher) or to ACTB RNA using the TaqMan primer probe set Mm02619580_g1 (Thermo Fisher). PCSK9 RNA expression is shown in the table below as the percentage of PCSK9 RNA (%UTC) relative to the amount of PCSK9 RNA in untreated control cells. [Table 97] [Table 98]

Claims

1. A polypeptide comprising a region having at least 800, at least 900, at least 1000, or at least 1050 linked amino acids, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity with any equal-length portion of SEQ ID NOs. 4-23 or 231-323, and optionally, the polypeptide is isolated.

2. A polypeptide comprising a region having at least 900, at least 1000, or at least 1100 linked amino acids, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity with the isolength portions of SEQ ID NOs. 6-23, 250-308, and optionally, the polypeptide is isolated.

3. A polypeptide comprising a region having at least 900, at least 1000, at least 1100, at least 1200, or at least 1300 linked amino acids, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity with any equal-length portion of SEQ ID NOs. 10-23 or 266-308, and optionally, the polypeptide is isolated.

4. A polypeptide comprising a region, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity with any of SEQ ID NOs: 4-23 or 231-323, and optionally, the polypeptide is isolated.

5. A polypeptide wherein the amino acid sequence of the polypeptide is at least 85%, at least 90%, at least 95%, or 100% identical to any of SEQ ID NOs: 4-23 or 231-323, and the polypeptide is optionally isolated.

6. The polypeptide according to claim 5, wherein the amino acid sequence of the polypeptide has at least 85%, at least 90%, at least 95%, or 100% identity with any of SEQ ID NOs: 12, 13, 20, 241, 242, 245, 247, 260, 266, 267, 268, 271, 274, 275, 279, 282, 283, 284, 288, 289, 292, 297, 299, 300, 301, 302, 314, or 323.

7. A polypeptide according to any one of claims 1 to 4, comprising the aforementioned region.

8. A polypeptide according to any one of claims 1 to 4, comprising heterogeneous domains.

9. The polypeptide according to claim 8, wherein the heterogeneous domain is selected from a transcription activator, a transcription repressor, a methyltransferase, a demethylase, a deaminase, an acetyltransferase, or a deacetylase.

10. The polypeptide according to any one of claims 1 to 4 or 7 to 9, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity with any equal-length portion of SEQ ID NOs: 13, 241, 267, 275, 284, 289, or 314.

11. The polypeptide according to any one of claims 1 to 10, wherein the polypeptide comprises a non-PI region and a PI domain, and the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs. 3186, 3188, 3190, 3192, 3194, 3196, or 3198.

12. The polypeptide according to any one of claims 1 to 10, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3186, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3187.

13. The polypeptide according to any one of claims 1 to 10, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3188, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3189.

14. The polypeptide according to any one of claims 1 to 10, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3190, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3191.

15. The polypeptide according to any one of claims 1 to 10, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3192, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3193.

16. The polypeptide according to any one of claims 1 to 10, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3194, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3195.

17. The polypeptide according to any one of claims 1 to 10, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3196, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3197.

18. The polypeptide according to any one of claims 1 to 10, wherein the polypeptide comprises a non-PI region and a PI domain, the non-PI region of the polypeptide has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3198, and the PI domain has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3199.

19. The polypeptide according to any one of claims 1 to 18, wherein the polypeptide has exactly two catalytically active nuclease active sites.

20. The polypeptide according to any one of claims 1 to 18, wherein the polypeptide has exactly one catalytically active nuclease active site.

21. The polypeptide according to any one of claims 1 to 18, wherein the polypeptide has zero catalytically active nuclease active sites.

22. A polypeptide according to any one of claims 1 to 21, comprising a nuclear localization signal.

23. The polypeptide according to claim 22, wherein the nuclear localization signal is located at the N-terminus of the polypeptide.

24. The polypeptide according to claim 22, wherein the nuclear localization signal is located at the C-terminus of the polypeptide.

25. The polypeptide according to claim 22, wherein the polypeptide comprises one or two nuclear localization signals at the N-terminus and C-terminus, respectively.

26. A nucleic acid encoding a polypeptide according to any one of claims 1 to 25.

27. The nucleic acid according to claim 26, wherein the nucleic acid is DNA.

28. The nucleic acid according to claim 26, wherein the nucleic acid is mRNA containing a coding region encoding a polypeptide according to any one of claims 1 to 25.

29. The nucleic acid according to claim 26, wherein the mRNA is an exogenous mRNA comprising a coding region encoding a polypeptide according to any one of claims 1 to 21.

30. mRNA according to claim 28 or 29, comprising a 5' cap structure.

31. mRNA according to any one of claims 28 to 30, comprising a 3'-poly(A) tail.

32. mRNA according to any one of claims 28 to 31, comprising 5'-UTR.

33. mRNA according to any one of claims 28 to 32, comprising 3'-UTR.

34. mRNA according to claim 28 or 29, comprising a 5'-cap structure and a 3'-poly(A) tail.

35. The mRNA according to any one of claims 28 to 34, wherein the coding region is codon-optimized for expression in eukaryotic cells.

36. The mRNA according to claim 35, wherein the coding region is codon-optimized for expression in mammalian cells.

37. The mRNA according to claim 36, wherein the mammalian cell is a human cell.

38. The mRNA according to any one of claims 28 to 37, wherein each nucleic acid base of the mRNA is selected from adenine, guanine, cytosine, uracil, thymine, and N1-methylpseudolacil.

39. The mRNA according to any one of claims 28 to 37, wherein each nucleic acid base of the mRNA is selected from adenine, guanine, cytosine, and N1-methylpseudracil.

40. The mRNA according to any one of claims 28 to 37, wherein each nucleic acid base of the mRNA is selected from adenine, guanine, cytosine, and uracil.

41. A guide, wherein the guide includes a protein recognition region, and the protein recognition region interacts with a polypeptide according to any one of claims 1 to 25.

42. The guide according to claim 41, wherein the guide is a dual guide.

43. The guide according to claim 41, wherein the guide is a single guide.

44. This is a guide, and the formula is as follows: T １ -D-X-S １ａ -B １ -S １ｂ -H １ -S １ｂ ’-B ２ -S １ａ ’-L １ -S ２ -H ２ -S ２ ’-L ２ -S ３ -H ３ -S ３ ’-(L ３ -S ４ -H ４ -S ４ ’) ｎ -(L ４ -S ５ -H ５ -S ５ ’) ｍ -T ２ It consists of oligonucleotides that follow the formula, In the array, D consists of 17 to 25 linked nucleosides, and the nucleic acid base sequence of D is complementary to the nucleic acid base sequence of the target DNA. X consists of one or two bound nucleosides that are absent or not complementary to the target DNA sequence. T １ and T ２ Each of these is either absent or independently consists of 1 to 30 bonded nucleosides. S １ａ and S １ａ Each of these consists of 6 to 10 bonded nucleosides, S １ａ The nucleic acid base sequence is S １ａ It is 100% complementary to the nucleic acid base sequence of ', S １ｂ and S １ｂ Each of these consists of 2 to 14 bonded nucleosides, S １ｂ The nucleic acid base sequence is S １ｂ It is 100% complementary to the nucleic acid base sequence of ', S ２ and S ２ Each of these consists of 2 to 6 bonded nucleosides, S ２ The nucleic acid base sequence is S ２ It is 100% complementary to the nucleic acid base sequence of ', S ３ and S ３ Each of these consists of 2 to 12 bonded nucleosides, S ３ The nucleic acid base sequence is S ３ It is 100% complementary to the nucleic acid base sequence of ', S ４ and S ４ Each of these consists of 4 to 14 bonded nucleosides, S ４ The nucleic acid base sequence is S ４ It is 100% complementary to the nucleic acid base sequence of ', S ５ and S ５ Each of these consists of 4 to 11 bonded nucleosides, S ５ The nucleic acid base sequence is S ５ It is 100% complementary to the nucleic acid base sequence of ', n is 0 or 1, m is 0 or 1, B １ However, it is either absent or consists of 1 to 3 bonded nucleosides. B ２ However, it is either absent or consists of 1 to 4 bonded nucleosides. B １ and B ２ Except when both are absent, B １ However, B ２ Not equal to, H １ , H ２ , H ３ , H ４ , and H ５ Each of these independently consists of 3 to 6 bonded nucleosides. L １ However, it consists of two to three bonded nucleosides, L ２ , L ３ , and L ４ Each of these is an independent guide consisting of 0 to 10 bonded nucleosides.

45. The guide according to claim 44, wherein the guide includes a protein recognition region that binds to the polypeptide described in any one of claims 1 to 25.

46. T １ However, the guide described in claim 44 or 45 is absent.

47. T ２ However, the guide described in any one of claims 44 to 46 is absent.

48. T ２ The guide according to any one of claims 44 to 46, wherein it comprises two to six bonded nucleosides containing uracil nucleic acid bases.

49. The guide according to any one of claims 44 to 48, wherein X is absent.

50. The guide according to any one of claims 44 to 49, wherein n is 1 and m is 0.

51. The guide according to any one of claims 44 to 50, wherein both n and m are 1.

52. B ２ The number of nucleosides in B １ A guide according to any one of claims 44 to 51, wherein the number of nucleosides is greater than the number of nucleosides in the guide.

53. Each L １ The guide according to any one of claims 44 to 52, wherein the nucleoside is adenosine.

54. The guide according to any one of claims 44 to 53, wherein the guide is bound to the polypeptide according to any one of claims 1 to 25.

55. T １ And X is absent, T ２ However, it is either absent or consists of 2 to 6 linked uridines. S １ａ and S １ａ Each of these consists of seven bonded nucleosides. S １ｂ and S １ｂ Each of these consists of 3 to 9 bonded nucleosides. S ２ and S ２ Each of these consists of three bonded nucleosides. S ３ and S ３ Each of these consists of four bonded nucleosides. S ４ and S ４ Each of these consists of five bonded nucleosides. S ５ and S ５ If present, each consists of 7 to 9 bonded nucleosides. n is 1, m is 0 or 1, B １ However, it consists of one nucleoside, B ２ consists of four linked nucleosides, H １ , H ２ , H ３ and H ４ Each of these, if present, consists of four bonded nucleosides. L １ However, it consists of two bonded adenosine nucleosides, L ２ However, it consists of five bonded nucleosides, L ３ consists of 0 nucleosides, L ４ The guide according to claim 44 or 45, wherein, if present, it consists of four bonded nucleosides.

56. S １ｂ and S １ｂ The guide according to claim 55, wherein each of the seven bonded nucleosides is comprised of seven bonded nucleosides.

57. S ５ and S ５ The guide according to claim 55 or 56, wherein each of the seven bonded nucleosides.

58. B ２ The guide according to any one of claims 55 to 57, wherein the nucleic acid base sequence is AAAG.

59. L ２ The guide according to any one of claims 55 to 58, wherein the nucleic acid base sequence is AAACU.

60. L ３ The guide according to any one of claims 55 to 59, wherein the nucleic acid base sequence is UUUUAA.

61. H ２ The guide according to any one of claims 55 to 60, wherein the nucleic acid base sequence is GUCA.

62. The guide according to any one of claims 55 to 61, wherein n is 1 and m is 0.

63. The guide according to any one of claims 55 to 62, wherein both n and m are 1.

64. S １ｂ and S １ｂ Each of these consists of seven bonded nucleosides, where n is 1 and m is 0, B ２ The nucleic acid base sequence is AAAG, L ２ The nucleic acid base sequence is AAACU, L ３ The nucleic acid base sequence is UUUUAA, H ２ The guide according to claim 55, wherein the nucleic acid base sequence is GUCA.

65. The guide according to any one of claims 55 to 64, wherein the guide comprises at least 40, at least 50, at least 60, or at least 70 nucleoside regions, and the nucleic acid base sequence of the region has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any isolength region of SEQ ID NOs: 131, 961-964, 1016-1020, 1052-1070, 1313-1314, or 2825.

66. The guide according to claim 65, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NOs: 131, 961-964, 1016-1020, 1052-1070, 1313-1314, or 2825.

67. The guide according to any one of claims 55 to 66, wherein D consists of 20 to 23 nucleosides having a nucleic acid base sequence that is 100% complementary to the nucleic acid base sequence of the target DNA.

68. T １ And X is absent, T ２ However, it is either absent or consists of six linked uridines. S １ａ and S １ａ Each of these consists of nine bonded nucleosides. S １ｂ and S １ｂ Each of these consists of 2 to 8 ink-coated nucleosides. S ２ and S ２ Each of these consists of six bonded nucleosides. S ３ and S ３ Each of these consists of seven bonded nucleosides. n and m are 0, B １ However, it consists of three nucleosides, B ２ However, it consists of four bonded nucleosides, H １ However, it consists of four bonded nucleosides, H ２ However, it consists of five bonded nucleosides, H ３ However, it consists of four bonded nucleosides, L １ However, it consists of two bonded adenosine nucleosides, L ２ The guide according to claim 44 or 45, wherein it consists of zero bound nucleosides.

69. S １ｂ and S １ｂ The guide according to claim 68, wherein each of the four bonded nucleosides is comprised of four bonded nucleosides.

70. L ２ However, it consists of 0 bonded nucleosides, T ２ The guide according to claim 68 or 69, comprising six bound uridines.

71. B ２ The guide according to any one of claims 68 to 70, wherein the nucleic acid base sequence is UAAC.

72. L ２ The guide according to any one of claims 68 to 71, wherein the nucleic acid base sequence is GAACUC.

73. H ２ The guide according to any one of claims 68 to 72, wherein the nucleic acid base sequence is UUUAU.

74. S １ｂ and S １ｂ Each of these consists of four bonded nucleosides, where n and m are 0, and B ２ The nucleic acid base sequence is UACC, L ２ The nucleic acid base sequence is GAACUC, H ２ The guide according to claim 68, wherein the nucleic acid base sequence is UUUAU.

75. The guide according to any one of claims 68 to 74, wherein the guide includes a region having a nucleic acid base sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of sequence numbers 520, 965-968, 1071-1094, or 1315-1318, and the region includes at least 40, at least 50, at least 60, or at least 70 nucleosides.

76. The guide according to claim 75, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NOs. 520, 965-968, 1071-1094, or 1315-1318.

77. The guide according to any one of claims 68 to 75, wherein D consists of 20 to 23 nucleosides having a nucleic acid base sequence that is 100% complementary to the nucleic acid base sequence of the target DNA.

78. T １ And X is absent, T ２ However, it is either absent or consists of 1 to 7 bonded nucleosides. S １ａ and S １ａ Each of these consists of seven bonded nucleosides. S １ｂ and S １ｂ Each of these consists of 4 to 12 bonded nucleosides. S ２ and S ２ Each of these consists of five bonded nucleosides. S ３ and S ３ Each of these consists of six bonded nucleosides. S ４ and S ４ Each of these consists of six bonded nucleosides. S ５ and S ５ If present, each consists of seven bonded nucleosides. n is 1, m is 0 or 1, B １ However, it consists of one nucleoside, B ２ However, it consists of three bonded nucleosides, H １ , H ２ , and H ３ Each of these consists of four bonded nucleosides, H ４ and H ５ However, if present, it consists of three bonded nucleosides. L １ However, it consists of three linked adenosine nucleosides, L ２ and L ３ However, it consists of zero bonded nucleosides. L ４ The guide according to claim 44 or 45, wherein, if present, it consists of 1 to 7 bonded nucleosides.

79. S １ｂ and S １ｂ The guide according to claim 78, wherein each of the six bonded nucleosides is comprised of six bonded nucleosides.

80. B ２ The guide according to any one of claims 78 to 79, wherein the nucleic acid base sequence is GAG.

81. H ２ The guide according to any one of claims 78 to 80, wherein the nucleic acid base sequence is AUCC.

82. S １ｂ and S １ｂ Each of these consists of six bonded nucleosides, where n is 1 and m is 0, B ２ The nucleic acid base sequence is GAG, H ２ The guide according to claim 78, wherein the nucleic acid base sequence is AUCC.

83. The guide according to any one of claims 78 to 82, wherein the guide includes a region having a nucleic acid base sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of sequence numbers 546, 973-976, 1021-1023, 1095-1126, or 1319-1342, and the region includes at least 40, at least 50, at least 60, or at least 70 nucleosides.

84. The guide according to claim 83, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NOs. 546, 973-976, 1021-1023, 1095-1126, or 1319-1342.

85. The guide according to any one of claims 78 to 84, wherein D consists of 20 to 24 nucleosides having a nucleic acid base sequence that is 100% complementary to the nucleic acid base sequence of the target DNA.

86. T １ And X is absent, T ２ However, it is either absent or consists of 1 to 6 bonded nucleosides. S １ａ and S １ａ Each of these consists of seven bonded nucleosides. S １ｂ and S １ｂ Each of these consists of 4 to 12 ink-coated nucleosides. S ２ and S ２ Each of these consists of four bonded nucleosides. S ３ and S ３ Each of these consists of four bonded nucleosides. S ４ and S ４ Each of these consists of five bonded nucleosides. S ５ and S ５ If present, each consists of seven bonded nucleosides. n is 1, m is 0 or 1, B １ However, it consists of one nucleoside, B ２ However, it consists of three nucleosides, H １ However, it consists of four bonded nucleosides, H ２ However, it consists of three bonded nucleosides, H ３ However, it consists of six bonded nucleosides, H ４ However, it consists of four bonded nucleosides, H ５ However, if present, it consists of three bonded nucleosides. L １ However, it consists of two bonded adenosine nucleosides, L ２ However, it consists of two bonded nucleosides, L ３ However, it consists of zero bonded nucleosides. L ４ The guide according to claim 44 or 45, wherein, if present, it consists of seven bonded nucleosides.

87. S １ｂ and S １ｂ The guide according to claim 86, wherein each of the ' ' consists of 10 bonded nucleosides.

88. The guide according to any one of claims 86 to 87, wherein n is 1 and m is 0.

89. B ２ The guide according to any one of claims 86 to 88, wherein the nucleic acid base sequence is GAG.

90. L ２ The guide according to any one of claims 86 to 89, wherein the nucleic acid base sequence is AA.

91. H ２ The guide according to any one of claims 86 to 90, wherein the nucleic acid base sequence is UAA.

92. S １ｂ and S １ｂ Each of these consists of 10 bonded nucleosides, where n is 1 and m is 0, B ２ The nucleic acid base sequence is GAG, L ２ The nucleic acid base sequence is AA, H ２ The guide according to claim 86, wherein the nucleic acid base sequence is UAA.

93. The guide according to any one of claims 86 to 92, wherein the guide includes a region having a nucleic acid base sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of sequence numbers 562, 985-989, 1027-1029, 1153-1182, or 1362-1390, and the region includes at least 40, at least 50, at least 60, or at least 70 nucleosides.

94. The guide according to claim 93, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NOs. 562, 985-989, 1027-1029, 1153-1182, or 1362-1390.

95. The guide according to any one of claims 86 to 94, wherein D consists of 20 to 24 nucleosides having a nucleic acid base sequence that is 100% complementary to the nucleic acid base sequence of the target DNA.

96. T １ And X is absent, T ２ However, it is either absent or consists of 2 to 5 bonded nucleosides. S １ａ and S １ａ Each of these consists of nine bonded nucleosides. S １ｂ and S １ｂ Each of these consists of 2 to 10 bonded nucleosides. S ２ and S ２ Each of these consists of four bonded nucleosides. S ３ and S ３ Each of these consists of nine bonded nucleosides. n and m are 0, B １ However, it consists of one nucleoside, B １ However, it consists of three nucleosides, H １ However, it consists of four bonded nucleosides, H ２ However, it consists of three bonded nucleosides, H ３ However, it consists of four bonded nucleosides, L １ However, it consists of two bonded adenosine nucleosides, L ２ The guide according to claim 44 or 45, comprising seven bonded nucleosides.

97. S １ｂ and S １ｂ The guide according to claim 96, wherein each of the six bonded nucleosides is comprised of six bonded nucleosides.

98. B ２ The guide according to claim 96 or 97, wherein the nucleic acid base sequence is CUA.

99. L ２ The guide according to any one of claims 96 to 98, wherein the nucleic acid base sequence is GUGUUUA.

100. H ２ The guide according to any one of claims 96 to 99, wherein the nucleic acid base sequence is AAA.

101. S １ｂ and S １ｂ Each of these consists of 10 bonded nucleosides, where n and m are 0, and B ２ The nucleic acid base sequence is CUA, L ２ The nucleic acid base sequence is GUGUUUA, H ２ The guide according to claim 96, wherein the nucleic acid base sequence is AAA.

102. The guide according to any one of claims 96 to 101, wherein the guide includes a region having a nucleic acid base sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of sequence numbers 593, 1006-1010, 1272-1292, 1394-1408, and 2827-2833, and the region includes at least 40, at least 50, at least 60, or at least 70 nucleosides.

103. The guide according to claim 102, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NOs. 593, 1006-1010, 1272-1292, 1394-1408, and 2827-2833.

104. The guide according to any one of claims 96 to 103, wherein D consists of 20 to 24 nucleosides having a nucleic acid base sequence that is 100% complementary to the nucleic acid base sequence of the target DNA.

105. T １ And X is absent, T ２ However, it is either absent or consists of three bonded nucleosides. S １ａ and S １ｂ But together, as well as S １ａ 'and S １ｂ 'These combine to form 12 to 20 bonded nucleosides, S ２ and S ２ Each of these consists of three bonded nucleosides. S ３ and S ３ Each of these consists of three bonded nucleosides. S ３ and S ３ Each of these consists of 10 to 13 bonded nucleosides. n is 1, and m is 0. B １ However, it consists of zero nucleosides. B ２ However, it consists of zero nucleosides. H １、 H ２ , H ３ , and H ４ However, it consists of four bonded nucleosides, L １ However, it consists of two bonded adenosine nucleosides, L ２ However, it consists of two bonded nucleosides, L ３ The guide according to claim 44 or 45, comprising six bonded nucleosides.

106. S １ａ and S １ｂ But together, as well as S １ａ 'and S １ｂ The guide according to claim 105, wherein the two groups together consist of 15 bonded nucleosides, each.

107. L ３ The guide according to claim 105 or 106, wherein the guide consists of three bonded nucleosides.

108. S ３ and S ３ The guide according to any one of claims 105 to 107, wherein each of the ' nucleosides consists of 13 bonded nucleosides.

109. L ２ The guide according to any one of claims 105 to 107, wherein the nucleic acid base sequence is GU.

110. H ２ The guide according to claim 108 or 109, wherein the nucleic acid base sequence is GAAA.

111. S １ａ and S １ｂ But together, as well as S １ａ 'and S １ｂ ', together, each consists of 15 bonded nucleosides, where n is 1 and m is 0, L ２ The nucleic acid base sequence is GU, H ２ The guide according to claim 105, wherein the nucleic acid base sequence is GAAA.

112. The guide according to any one of claims 105 to 111, wherein the guide includes a region having a nucleic acid base sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of sequence numbers 568, 990-993, 1030-1034, 1183-1206, or 1391-1393, and the region includes at least 40, at least 50, at least 60, or at least 70 nucleosides.

113. The guide according to claim 112, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NO: 568, 990-993, 1030-1034, 1183-1206, or 1391-1393.

114. The guide according to any one of claims 106 to 113, wherein D consists of 20 to 24 nucleosides having a nucleic acid base sequence that is 100% complementary to the nucleic acid base sequence of the target DNA.

115. This is a guide, and the formula is as follows: T １ -D-X-S １ａ -B １ -S １ｂ -H １ -S １ｂ ’-B ２ -S １ａ ’-L １ -S ２ -H ２ -S ２ ’-L ２ -S ３ -H ３ -S ３ ’-(L ３ -S ４ -H ４ -S ４ ’) ｎ -T ２ It consists of oligonucleotides that follow the formula, In the array, D consists of 17 to 25 linked nucleosides, and the nucleic acid base sequence of D is complementary to the nucleic acid base sequence of the target DNA. X is either absent or consists of one or two bound nucleosides that are not complementary to the nucleic acid base sequence of the target DNA. T １ and T ２ Each of these is either absent or independently consists of 1 to 30 bonded nucleosides. S １ａ and S １ａ Each of these consists of seven bonded nucleosides, S １ａ The nucleic acid base sequence is S １ａ It is 100% complementary to the nucleic acid base sequence of ', S １ｂ and S １ｂ Each of these consists of 4 to 12 bonded nucleosides, S １ｂ The nucleic acid base sequence is S １ｂ It is 100% complementary to the nucleic acid base sequence of ', S ２ and S ２ Each of these consists of six bonded nucleosides, S ２ The nucleic acid base sequence is S ２ It is 100% complementary to the nucleic acid base sequence of ', S ３ and S ３ Each of these consists of five or six bonded nucleosides, S ３ The nucleic acid base sequence is S ３ It is 100% complementary to the nucleic acid base sequence of ', S ４ and S ４ If present, each consists of six bonded nucleosides, S ４ The nucleic acid base sequence is S ４ It is 100% complementary to the nucleic acid base sequence of ', n is 0 or 1, B １ However, it consists of one nucleoside, B ２ However, it consists of three bonded nucleosides, H １ , H ２ , H ３ , and H ４ Each of these independently consists of 3 to 4 bonded nucleosides. L １ However, it consists of 17 bonded nucleosides, L ２ However, it consists of zero bonded nucleosides. L ３ However, the guide consists of eight bonded nucleosides.

116. S １ｂ and S １ｂ The guide according to claim 115, wherein each of the eight bonded nucleosides is comprised of eight bonded nucleosides.

117. B ２ The guide according to claim 115 or 116, wherein the nucleic acid base sequence is GAA.

118. L ２ The guide according to any one of claims 115 to 117, wherein the nucleic acid base sequence is AAAAAAAUUUAUUCAAAAC (Sequence ID 78).

119. H ２ The guide according to any one of claims 115 to 118, wherein the nucleic acid base sequence is GAAA.

120. S １ｂ and S １ｂ Each of these consists of eight bonded nucleosides, where n and m are 0, and B ２ The nucleic acid base sequence is GAA, L ２ The nucleic acid base sequence is AAAAAAAUUUAUUCAAAAC (Sequence ID 78), H ２ The guide according to claim 119, wherein the nucleic acid base sequence is GAAA.

121. The guide according to any one of claims 115 to 120, wherein the guide includes a region having a nucleic acid base sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of sequence numbers 554, 981-984, 1024-1026, 1127-1152, or 1343-1361, and the region includes at least 40, at least 50, at least 60, or at least 70 nucleosides.

122. The guide according to claim 121, wherein the nucleic acid base sequence of the region has 100% identity with any of the isolength regions of SEQ ID NOs. 554, 981-984, 1024-1026, 1127-1152, or 1343-1361.

123. The guide according to any one of claims 115 to 122, wherein D consists of 20 to 24 nucleosides having a nucleic acid base sequence that is 100% complementary to the nucleic acid base sequence of the target DNA.

124. The guide according to any one of claims 41 to 43, wherein the guide includes a region having a nucleic acid base sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of sequence numbers 121 to 141, 510 to 602, 961 to 1430, or 1436 to 2833, and the region includes at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 nucleosides.

125. The guide according to any one of claims 41 to 43, wherein the guide includes a protein recognition region having a nucleic acid base sequence that is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of sequence numbers 121 to 141, 510 to 602, 961 to 1430, and 1436 to 2833.

126. The guide according to any one of claims 41 to 125, wherein the guide comprises a modified oligonucleotide.

127. The guide according to any one of claims 41 to 126, wherein the guide comprises at least one modified sugar moiety.

128. The guide according to claim 127, wherein the modified sugar portion is selected from 2'-OMe and 2'-F.

129. The guide according to claim 127, wherein the modified oligonucleotide contains a 2'-OMe or 2'-F sugar moiety within the first five nucleosides at the 5' end of the guide or within the last five nucleosides at the 3' end of the guide.

130. The guide according to claims 127 to 129, wherein the guide includes two to three 2'-OMe sugar moieties at its 5' end and two to three 2'-OMe sugar moieties at its 3' end.

131. The guide according to claim 130, wherein each non-2'-OMe sugar portion of the guide includes an unmodified RNA sugar portion.

132. The guide according to any one of claims 127 to 131, wherein the guide includes a modified nucleoside bond.

133. The guide according to claim 132, wherein the modified nucleoside bond is a phosphorothioate nucleoside bond.

134. The guide according to claim 132 or 133, wherein the guide includes at least one modified internucleoside bond within the first five nucleosides at the 5' end of the guide or within the last five nucleosides at the 3' end of the guide.

135. The guide according to claim 134, wherein the guide includes two to three phosphorothioate nucleoside bonds at its 5' end and two to three phosphorothioate nucleoside bonds at its 3' end.

136. The guide according to claim 135, wherein each remaining internucleoside bond is an unmodified phosphodiester internucleoside bond.

137. The guide according to any one of claims 132 to 136, wherein each nucleoside bond in the guide is a standard-length nucleoside bond.

138. The guide according to any one of claims 41 to 125, wherein the guide comprises an unmodified oligonucleotide.

139. The guide according to any one of claims 41 to 138, wherein the guide includes a target recognition region that is at least 90%, at least 95%, or 100% complementary to the target DNA sequence.

140. The guide according to claim 139, wherein the target recognition region comprises 17-25, 19-24, 20-24, 20-23, 20-22, 21-22, 21-23, 21-24, 20, 21, 22, 23, or 24 bound nucleosides.

141. An editing system comprising a polypeptide according to any one of claims 1 to 25 and a guide according to any one of claims 41 to 139.

142. It is an editing system, A nucleic acid according to any one of claims 26 to 40, An editing system comprising a guide according to any one of claims 41 to 140.

143. It is an editing system, A nucleic acid according to any one of claims 26 to 40, An editing system comprising a nucleic acid encoding a guide according to any one of claims 41 to 125 or 138.

144. The editing system according to claim 142 or 143, wherein the nucleic acid encoding the polypeptide is exogenous mRNA.

145. The editing system according to claim 142 or 143, wherein the nucleic acid encoding the polypeptide is DNA.

146. The editing system according to any one of claims 143 to 145, wherein the nucleic acid encoding the guide is DNA.

147. A composition comprising an editing system according to any one of claims 141 to 146 and an LNP.

148. A viral vector comprising the editing system described in claim 143.

149. A method for editing a target nucleic acid, comprising administering the composition according to claim 146 or the viral vector according to claim 148 to the target.

150. A method for editing a target nucleic acid, comprising contacting a cell with the composition according to claim 147 or the viral vector according to claim 148.

151. A method for forming double-strand breaks or nicks in a target nucleic acid, comprising contacting a cell with the composition according to claim 147 or the viral vector according to claim 148.

152. A composition according to claim 147 or a viral vector according to claim 148 for use in therapy.

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